Methods of determining stabilization compounds for predictive biomarkers

ABSTRACT

Methods of determining stabilization compounds for predictive biomarkers. According to at least one embodiment of a method of the present disclosure, the method comprises introducing a diagnostic marker and a stabilization agent to a detection platform, mixing the diagnostic marker and stabilization agent, and determining their binding characteristics. Additionally, the method may further comprise generating a report based on the binding characteristics and delivering the report to a recipient.

PRIORITY

This U.S. utility patent application claims the priority benefit of U.S.Provisional Patent Application Ser. No. 61/390,002, filed Oct. 5, 2010;U.S. Provisional Patent Application Ser. No. 61/385,347, filed Sep. 22,2010; U.S. Provisional Patent Application Ser. No. 61/383,401, filedSep. 16, 2010; U.S. Provisional Patent Application Ser. No. 61/379,598,filed Sep. 2, 2010; U.S. Provisional Patent Application Ser. No.61/378,960, filed Sep. 1, 2010; U.S. Provisional Patent Application Ser.No. 61/373,619, filed Aug. 13, 2010; U.S. Provisional Patent ApplicationSer. No. 61/364,964, filed Jul. 16, 2010; U.S. Provisional PatentApplication Ser. No. 61/364,969, filed Jul. 16, 2010; U.S. ProvisionalPatent Application Ser. No. 61/364,975, filed Jul. 16, 2010; U.S.Provisional Patent Application Ser. No. 61/364,978, filed Jul. 16, 2010;U.S. Provisional Patent Application Ser. No. 61/364,982, filed Jul. 16,2010; U.S. Provisional Patent Application Ser. No. 61/365,179, filedJul. 16, 2010; U.S. Provisional Patent Application Ser. No. 61/322,768,filed Apr. 9, 2010; and U.S. Provisional Patent Application Ser. No.61/303,165, filed Feb. 10, 2010. The contents of each of theseapplications are hereby incorporated by reference in their entirety intothis disclosure.

BACKGROUND

Biomarkers are molecular indicators of a specific biological property, abiochemical feature or facet that can be used to measure the progress ofa disease or the effects of a treatment. For example, serum low-densitylipoprotein (LDL) is a biomarker of cholesterol and blood pressure,while the P53 gene is a biomarker for cancer. For chronic diseases andconditions, such as diabetes and allergies, accurate diagnosis isparticularly important, especially where the side effects of a treatmentare severe.

Diagnostic tests using biomarkers as molecular indicators not onlydetect the presence or absence of the biomarker, but often must measurethe exact concentration of a biomarker to determine whether an abnormalcondition exists. Because of the requirement for accuracy, the processof sample collection, preparation, and analysis are often complicatedand time consuming. Currently, blood-based assays for biomarker presenceor activity are considered to be the “gold standard” for biomarker-typeassays.

Despite the desire for accurate results, rapid, point of care analysisof biological samples using biomarkers is becoming increasinglyimportant in the present medical environment due to the need for quickresults.

SUMMARY

The disclosure of the present application provides methods for theanalysis of stabilizing agents.

In at least one embodiment of a method for analyzing stabilizing agentsof the present disclosure, the method comprises the steps of introducinga diagnostic marker into each of a plurality of detection sites of adetection platform, where the detection platform comprises the pluralityof detection sites each capable of receiving a diagnostic marker, astabilization agent, and a detection agent, a detection device capableof determining a binding characteristic between the detection agent andthe diagnostic marker in the detection sites, a computer databasecapable of receiving a plurality of binding characteristics, theplurality of binding characteristics comprising at least one bindingproperty of a diagnostic marker to a detection agent, and a processoroperably coupled to the computer database and the detection device. Theprocessor, in at least one embodiment, has and executes a softwareprogram operational to determine the binding characteristic of thedetection agent to the diagnostic marker in each of the plurality ofdetection sites, compare the binding characteristic among each of theplurality of detection sites to order the level of effect of thestabilizing agents assayed on the binding characteristic between thedetection agent and the diagnostic agent, generate a binding recordusing the compared binding characteristics, and deliver the bindingrecord to a recipient. At least one embodiment of the method of thepresent disclosure also includes the steps of introducing thestabilization agent to each of the plurality of detection sitescontaining the diagnostic marker, combining the detection agent with thestabilization agent and diagnostic marker in the detection sites,comparing the binding characteristic with the processor among each ofthe plurality of detection sites to order the level of effect of thestabilizing agents assayed on the binding characteristic between thedetection agent and the diagnostic agent, generating a binding reportusing the compared binding characteristics, and delivering the bindingreport to a recipient.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the stabilizing agent is selected fromthe group consisting of a protease inhibitor, a DNase inhibitor, and aRNase inhibitor. Further, the stabilizing agent, in at least oneembodiment, may prevent the degradation or inactivation of thediagnostic marker for at least a twenty four hours period followinginteraction with the diagnostic agent.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the diagnostic marker is selected fromthe group consisting of a protein, a glycoprotein, a nucleic acid, anenzyme, an enzyme inhibitor, and a metabolite.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the stabilizing agent is useful tocompletely or substantially inactivate an enzyme selected from the groupconsisting of an amylase, a lysozyme, a peroxidase, a glycosidase, anesterase, a protease, and a peptidase. Further, the stabilization agentin at least one embodiment may be selected from the group consisting ofFixanal® Buffer 6.0 (Sigma-Aldrich Co.), acetic acid, aluminum hydroxidebentonite, aluminum sulfate hydrate, aluminum potassium sulfatedodecahydrate, benzoic acid, caffeine, and 3-tert-butyl-hydroxyanisole,or a combination thereof.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the stabilizing agent comprises aplurality of stabilizing agents each present in approximately the sameconcentration. The stabilizing agent may also be capable in at least oneembodiment of inhibiting degradation of the diagnostic marker to aninhibitory degree, wherein the inhibitory degree is selected from thegroup consisting of at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, and at least about99%.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the stabilizing agent has aconcentration selected from the group consisting of about 200 parts permillion (ppm) to about 2000 ppm, about 400 ppm to about 1600 ppm, about600 ppm to about 1400 ppm, about 800 ppm to about 1200 ppm, and about400 ppm to about 600 ppm.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the stabilizing agent is able toinhibit the degradation or inactivation of the diagnostic marker for aninhibitory period selected from the group consisting of at least oneminute, at least about five minutes, at least about ten minutes, atleast about fifteen minutes, at least about thirty minutes, at leastabout one hour, at least about two hours, at least about four hours, andat least about eight hours.

In at least one embodiment of the method of analyzing stabilizationagents of the present disclosure, the diagnostic marker is selected fromthe group consisting of Aldose reductase, Angiogenin, Annexin A1, B-cellactivating factor (BAFF), B-cell lymphoma 2 (BCL2)-like 2, Beta Humanchorionic gonadotropin, Ca15-3, Calcyclin, Calvasculin, Cancer AntigenCA 19-9, Cancer Antigen CA 15-3, Cathepsin D, Caveolin-1, ChromograninA, Alpha-crystallin B chain (CRYAB), Endostatin, Eotaxin-2, Epithelialcell adhesion molecule (EpCAM), Ezrin, fatty acid binding protein 4(FABP4), Galectin-3, γ-glutamylcysteine ligase regulatory chain (GCLR),Gelsolin, Glucose 6-phosphate (G6P), Glycoprotein 130 (gp130),Glutathione S-transferase Mu 1 (GSTM1), Hepsin, High-mobility groupprotein B1 (HMGB-1), Insulin-like growth factor binding protein 1(IGFBP-1), Insulin-like growth factor binding protein 4 (IGFBP-4),Insulin-like growth factor binding protein 5 (IGFBP-5), Insulin-likegrowth factor binding protein 6 (IGFBP-6), LGL, latency associatedpeptide (LAP), macrophage stimulating protein (MSP), MHC class Ipolypeptide-related sequence A (MICA), Nucleoside diphosphate kinase B(NME2), Neuron-specific Enolase (NSE), Osteopontin, Osteoprotegerin,Pepsinogen, Peroxiredoxin, Phosphoserine aminotransferase (PSAT1),Prostate Specific Antigen, Receptor tyrosine-protein kinase erbB-3(ErbB3), Serpin B3, Vascular smooth muscle cell growth factor R2 (VSGFR2/KDR), Vascular endothelial growth factor R3 (VEGF R3/Flt-4),Thyroglobulin, Tyrosine kinase with immunoglobulin-like and EGF-likedomains 2 (TIE-2), Tissue plasminogen activator (tPA), Transforminggrowth factor beta (TGF-β1), Tumor necrosis factor receptor 1 (TNF-R1),urokinase-type Plasminogen Activator (uPA), urokinase-type PlasminogenActivator Receptor (uPAR), BrcaI, BrcaII, kallikreins, e-cadherin, Hoxpeptide, and Engrailed-2.

DESCRIPTION OF THE DRAWINGS

The features and advantages of the present disclosure, and the manner ofattaining them, will be more apparent and better understood by referenceto the following descriptions taken in conjunction with the accompanyingfigures, wherein:

FIGS. 1A and B show a diagram of a detection system, according to atleast one embodiment of the present disclosure;

FIGS. 2-4 shows a diagram of a method of producing a bead system,according to at least one embodiment of the present disclosure;

FIG. 5 shows a flowchart of a method for creating a bead system,according to at least one embodiment of the present disclosure;

FIGS. 6A and B show a diagram of a test strip, according to at least oneembodiment of the present disclosure;

FIG. 6C show a diagram of a microarray, according to at least oneembodiment of the present disclosure;

FIG. 6D shows a diagram of a microfluidic system, according to at leastone embodiment of the present disclosure;

FIG. 7 shows a flowchart of a method of stabilizing diagnostic markers,according to at least one embodiment of the present disclosure;

FIG. 8 shows a flowchart of a method analyzing a diagnostic markers,according to at least one embodiment of the present disclosure;

FIG. 9 shows a flowchart of a method of improving biomarkeravailability, according to at least one embodiment of the presentdisclosure;

FIG. 10 shows a flowchart of a method of identifying diagnostic markersof a disease state, according to at least one embodiment of the presentdisclosure;

FIG. 11 shows a flowchart of a method for screening biomarkers,according to at least one embodiment of the present disclosure;

FIG. 12 shows a flowchart of a method of stabilizing diagnostic markers,according to at least one embodiment of the present disclosure;

FIG. 13 shows a an exemplary system framework, according to at least oneembodiment of the present disclosure;

FIG. 14 shows a graphical depiction of α-amylase inhibition according toat least one embodiment of the present disclosure;

FIG. 15 shows a graphical depiction of α-amylase inhibition according toat least one embodiment of the present disclosure;

FIG. 16 shows a graphical depiction of lysozyme inhibition according toat least one embodiment of the present disclosure;

FIG. 17 shows a graphical depiction of a galactose oxidase screenaccording to at least one embodiment of the present disclosure;

FIG. 18A shows a three dimensional plot of glycated Hemoglobin A1c(HbA1c) analyzed by high pressure liquid chromatography (HPLC) accordingto at least one embodiment of the present disclosure;

FIG. 18B shows a ultraviolet (UV) image of HbA1c analyzed by HPLCaccording to at least one embodiment of the present disclosure;

FIG. 18C shows a chromatogram of HbA1c analyzed by HPLC according to atleast one embodiment of the present disclosure;

FIG. 19A shows a three dimensional plot of HbA1c analyzed by HPLCfollowing exposure to saliva according to at least one embodiment of thepresent disclosure;

FIG. 19B shows a UV image of HbA1c analyzed by HPLC following exposureto saliva according to at least one embodiment of the presentdisclosure;

FIG. 19C shows a chromatogram of HbA1c analyzed by HPLC followingexposure to saliva according to at least one embodiment of the presentdisclosure;

FIG. 20A shows a three dimensional plot of HbA1c analyzed by HPLCfollowing exposure to saliva and a stabilizing mixture according to atleast one embodiment of the present disclosure;

FIG. 20B shows a UV image of HbA1c analyzed by HPLC following exposureto saliva and a stabilizing mixture according to at least one embodimentof the present disclosure;

FIG. 20C shows a chromatogram of HbA1c analyzed by HPLC followingexposure to saliva and a stabilizing mixture according to at least oneembodiment of the present disclosure;

FIG. 21 shows a comparison of chromatograms from HPLC analyzed HbA1c(FIG. 21A), HbA1c+Saliva (FIG. 21B), and HbA1c+Saliva+Stabilizingmixture (FIG. 21C) according to at least one embodiment of the presentdisclosure;

FIG. 22 shows a comparison of UV images from HPLC analyzed HbA1c (FIG.22A), HbA1c+Saliva (FIG. 22B), and HbA1c+Saliva+Stabilizing mixture(FIG. 22C) according to at least one embodiment of the presentdisclosure;

FIG. 23 shows a comparison of three dimensional plots from HPLC analyzedHbA1c (FIG. 23A), HbA1c+Saliva (FIG. 23B), and HbA1c+Saliva+Stabilizingmixture (FIG. 23C) according to at least one embodiment of the presentdisclosure;

FIG. 24A shows a three dimensional plot of Cancer Antigen 19-9 (CA 19-9)analyzed by high pressure liquid chromatography (HPLC) according to atleast one embodiment of the present disclosure;

FIG. 24B shows a ultraviolet (UV) image of CA 19-9 analyzed by HPLCaccording to at least one embodiment of the present disclosure;

FIG. 25A shows a three dimensional plot of CA 19-9 analyzed by HPLCfollowing exposure to saliva according to at least one embodiment of thepresent disclosure;

FIG. 25B shows a UV image of CA 19-9 analyzed by HPLC following exposureto saliva according to at least one embodiment of the presentdisclosure;

FIG. 26A shows a three dimensional plot of CA 19-9 analyzed by HPLCfollowing exposure to saliva and a stabilizing mixture according to atleast one embodiment of the present disclosure;

FIG. 26B shows a UV image of CA 19-9 analyzed by HPLC following exposureto saliva and a stabilizing mixture according to at least one embodimentof the present disclosure;

FIG. 27 shows a comparison of three dimensional plots from HPLC analyzedCA 19-9 (FIG. 27A), CA 19-9+Saliva (FIG. 27B), and CA19-9+Saliva+Stabilizing mixture (FIG. 27C) according to at least oneembodiment of the present disclosure;

FIG. 28 shows a comparison of UV images from HPLC analyzed CA 19-9 (FIG.28A), CA 19-9+Saliva (FIG. 28B), and CA 19-9+Saliva+Stabilizing mixture(FIG. 28C) according to at least one embodiment of the presentdisclosure;

FIG. 29 shows a graphical depiction of Peroxidase activity inhibition byan inhibitor cocktail according to at least one embodiment of thepresent disclosure;

FIG. 30 shows a graphical depiction of Peroxidase activity inhibition bycaffeine according to at least one embodiment of the present disclosure;

FIG. 31 shows a graphical depiction of Peroxidase activity inhibition by3-tert-butyl-hydroxyanisole according to at least one embodiment of thepresent disclosure;

FIG. 32 shows a graphical depiction of Peroxidase activity inhibition bybenzoic acid according to at least one embodiment of the presentdisclosure;

FIG. 33 shows a graphical depiction of Peroxidase activity inhibition byaluminum potassium sulfate dodecahydrate according to at least oneembodiment of the present disclosure;

FIG. 34 shows a schematic of a diagnostic system, according to at leastone embodiment of the present disclosure;

FIG. 35 shows a graphical representation of an assay component,according to at least one embodiment of the present disclosure; and

FIG. 36 shows a graphical depiction of a fluid collection device,according to at least one embodiment of the present disclosure.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

The disclosure of the present application provides various compositions,systems, and methods for biomarker stabilization and analysis.Specifically, formulations are disclosed herein, which function topreserve the level of diagnostic markers, such as biomarkers, in bodyfluids, from enzymatic alteration, or degradation. Additionally, methodsfor the collection and analysis of body fluids are disclosed. Themethods and formulations disclosed herein can be used to improve thesampling and testing of a body fluid by conditioning the body fluids forthe stabilization of specific biomolecules and/or drug metabolites. Asused herein, the term “body fluids” includes fluids produced by thebody, such as saliva, or fractions thereof, mucous secretions, tears,sweat, bile, semen, urine, vaginal secretions, exhalations, analsecretions, blood, plasma, serum and mixtures of thereof. Body fluidsmay also comprise cancer cells, peripheral blood mononuclear cells,lymphocytes, lymph fluid, and other tissue secretions or fluid.

Saliva

Saliva is clear, viscous fluid with a slightly alkaline pH and a pIrange from 11.5-3.0. It is hypotonic, composed of about 99.5% water, andalso contains ions (e.g., K⁺, Na⁺, Ca²⁺, Mg²⁺, H⁺, Cl⁻, HCO₃ ⁻, F⁻, HPO₄²⁻), and small organic molecules (e.g., ureas, hormones, lipids, DNA,and RNA). There are multiple contributors to the composition of saliva.Saliva has a complex “proteome”-10⁶ D glycoproteins to 1000D peptides.It contains secretory products of salivary glands, products of B cells,PMNs, epithelial cells, and bacteria. Major (e.g., parotid,submandibular, and sublingual) and minor (e.g., palatine and retromolar)glands contribute to the composition of saliva, along with extraneouscontributors such as gingival crevicular fluid, serum proteins, whiteblood cells and their byproducts, oral epithelial cells, oral bacteria,food debris and dissolved food components.

Saliva from different glands may differ in composition. For example,saliva from the parotid gland is dominated by serous secretory cells,whereas saliva from the submandibular and sublingual glands and minorglands are mixed serous or mostly mucous. There can also be qualitativeand quantitative differences in saliva output that affect itscomposition. Glandular contribution to saliva is affected by level ofgland activity. The amount of saliva secreted per minute or the salivaryflow rate influences the concentration of the constituents as well asthe proportion of the constituents from each of the three pairs of majorsalivary glands and the minor salivary glands.

I. Stabilizing Agents

According to at least one embodiment of the present disclosure,conditioning of body fluids for the preservation of diagnostic markersmay be accomplished through the use of stabilizing agents, which may beincorporated into one or more carrier vehicles, such as rinses, gums,beverages, and confectionaries. For example, specific rinses and/orpre-rinses specially formulated according to the specific molecule ormolecules (the diagnostic marker) to be detected in the body fluid, maycontain a stabilization agent. The use of rinses and pre-rinses of thepresent disclosure to condition the body fluid may enhance thesensitivity for detection of specific diagnostic markers for clinicaldiagnosis. Additionally, this process may improve the signal-to-noiseratio for a better diagnostic yield. Further, each rinse/pre-rinse canbe specifically formulated to reduce or prevent false positive and falsenegative results.

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may act to prevent/decrease thedegradation, or reduction of activity, of diagnostic markers. In atleast one embodiment, the stabilizing agent may be comprised of one ormore Generally Regarded as Safe (GRAS) compound, as determined by theFood and Drug Administration of the United States of America, such asthose listed in Table I. An exemplary embodiment of a stabilizationagent may act to stabilize a diagnostic marker, and/or to bind to adestructive component to prevent the destructive component fromdegrading or decreasing the activity of the diagnostic marker.

In additional exemplary embodiments of the stabilization agent, thestabilizing agent may comprise at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, or at least 25 compounds which acts to stabilizethe diagnostic marker, and/or to bind to a destructive component toprevent the destructive component from degrading or decreasing theactivity of the diagnostic marker. Further, in various embodiments thestabilizing agent of the present disclosure may be present in at a levelof between about 200 ppm to about 2,000 ppm, about 400 ppm to about 1600ppm, about 600 ppm to about 1400 ppm, about 800 ppm to about 1200 ppm.Moreover, in at least one embodiment, the stabilizing agent may bepresent at a level of approximately 500 ppm.

TABLE I GRAS Compounds as Stabilizing Agents 1 Agar Sigma-Aldrich 2Alginic acid sodium salt Sigma-Aldrich 3 L-Ascorbic acid Sigma-Aldrich 4(+)-Sodium ascorbate Sigma-Aldrich 5 Buffer solution Fluka 6 Sodiumcitrate Sigma-Aldrich 7 Tragacanth Sigma-Aldrich 8 Gum arabic, fromacacia tree Sigma-Aldrich 9 Sodium tripolyphosphate Sigma-Aldrich 10Potassium citrate monobasic Sigma-Aldrich 11 Sodium pyrophosphatetetrabasic Sigma-Aldrich 12 Calcium citrate Sigma-Aldrich 13 Citric acidSigma-Aldrich 14 Potassium sodium tartrate tetrahydrate Sigma-Aldrich 15Sodium benzoate Sigma-Aldrich 16 Benzoic acid Sigma-Aldrich 17D-aspartic acid Sigma-Aldrich 18 Sodium phosphate dibasic Sigma-Aldrich19 Malic acid Sigma-Aldrich 20 Sodium propionate Sigma-Aldrich 21 SodiumL-ascorbate Sigma-Aldrich 22 DL-Phenylalanine Sigma-Aldrich 23 CalciumL-ascorbate dihydrate Sigma-Aldrich 24 Choline chloride Sigma-Aldrich 25L-Ascorbic acid Sigma-Aldrich 26 Sodium diacetate SAFC 27 PH buffer 4.01Orion 28 PH Buffer 7.00 Orion 29 PH buffer 10.0 Orion 30 Fixanal Buffer6.0 Sigma-Aldrich 31 Ammonium Chloride Sigma-Aldrich 32 Ammonium Citratetribasic, anhydrous Sigma-Aldrich 33 Caffeine Sigma-Aldrich 34 Aluminumsulfate hydrate Sigma-Aldrich 35 Beeswax refined Sigma-Aldrich 36Aluminum Hydroxide Sigma-Aldrich 37 Bentonite Sigma-Aldrich 38 Ammoniumbicarbonate Sigma-Aldrich 39 3-tert--butyl-hydroxyanisole Sigma-Aldrich40 Benzoic acid Sigma-Aldrich 41 trans-aconitic acid Sigma-Aldrich 42Aluminum potassium sulfate Sigma-Aldrich dodecahydrate 43 Adpic acidSigma-Aldrich 44 Ammonium hydrogen phosphate Sigma-Aldrich 45 Ammoniumdihydrogen phosphate Sigma-Aldrich 46 Ammonium carbonate Sigma-Aldrich47 Acetic acid Sigma-Aldrich

According to at least one exemplary embodiment, the stabilizing agentmay block at least one enzymatic activity of a destructive component.The destructive component may be one or more component of a bodily fluidwhich acts to degrade, or decrease the activity of a diagnostic marker.Further, the destructive component may be one or more Amylases,Lysozymes, Peroxidases, Glycosidases, Esterases, Proteases, and/orPeptidases. In an exemplary embodiment, the stabilizing agent may be anaturally occurring or artificial protease inhibitor, DNase inhibitor,or RNase inhibitor. Specifically, a natural stabilizing agent mayinclude any component found from food products (including, but notlimited to, the Solanaceae family), herbs, or spices that acts todecrease the effect of a destructive component. For example, exemplaryembodiments of the natural stabilizing agents may be any of theinhibitors found in lima beans, soybeans, or avian eggs (such asovomucoid glycoprotein protease inhibitors) (See Table II). Moreover, anembodiment of the stabilizing agent may also comprise a molecule whichspecifically inhibits the activity of a molecule which targets thediagnostic marker for degradation or inactivation. Further, thestabilizing agent may act to alter the composition of the body fluid(such as pH, or ion concentration) to decrease the degradation orinactivation of the diagnostic marker.

TABLE II Molecular Inhibitory Material weight power Details Lima beans8-10 kDa 2.2 times There are six different weight lima bean inhibitors.Ovomucoid 8-10 kDa 1.2 times Ovomucoids are the weight glycoproteinprotease inhibitors found in raw avian egg white. There are otherprotease inhibitors in ovomucoids as well. Soybeans 20.7-22.3 kDa    1.2 times Soybeans contain weight several inhibitors; the one in thechart is considered the primary one. All of them bind chymotrypsin to alesser degree.

As used herein, “diagnostic marker” may be any biological molecule whosepresence, specific concentration, or specific activity may be indicativeof a disease state or heath/lifestyle characteristic. As used herein,“indicative of a disease state” refers to the presence, progression,prevention, remission, amelioration, or prognosis of a disease. Thisalso includes, but is not limited to, the effects of a therapeutic on adisease. The disease state analyzed through use of the diagnostic markermay in an exemplary embodiment be directed towards any cancer, metabolicdisease (such as diabetes I or II), cardiovascular disease, or thepredisposition of any of the same. Additionally, a disease state mayalso include the infection of the patient by a bacterial, virus, yeast,fungus, or parasite.

A. Disease States

Bacterial infections, as referenced herein, may include a mammalianinfection by any bacterial species. Embodiments of the bacterialspecies, as used herein, may include, but are not limited to, Bacillusanthracis, Bacillus cereus, Staphylococcus aureus, Listeriamonocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Borrelia burgdorferi, Treponema pallidum, Chlamydiatrachomatis, Chlamydophila psittaci, Corynebacterium diphtherias,Mycobacterium tuberculosis, and Mycobacterium avium, Rickettsiaprowazekii, Rickettsia rickettsii, Rickettsia typhi, Anaplasmaphagocytophilum, Ehrlichia chaffeensis, Brucella melitensis, Bordetellapertussis, Burkholderia mallei, B. pseudomallei, Neisseria gonorrhoeae,Neisseria meningitides, Campylobacter jejuni, Helicobacter pylori,Legionella pneumophila, Acinetobacter baumannii, Moraxella catarrhalis,Pseudomonas aeruginosa, Aeromonas sp., Vibrio cholerae, Vibrioparahaemolyticus, Thiotrichales sp., Haemophilus influenzae, Klebsiellapneumoniae, Proteus mirabilis, Yersinia pestis, Yersinia enterocolitica,Shigella flexneri, Salmonella enterica, and Escherichia coli.

Viral infections, as referenced herein, may include a mammalianinfection by any viral species. Embodiments of the viral species, asused herein, may include, but are not limited to, single-stranded DNAviruses, including the genus Parvoviridae, double-stranded DNA viruses,including the genus Papillomaviridae, Polyomaviridae, Poxyiridae,Herpesviridae (ex. Herpes Simplex Virus 1, 2, 6, Varicella-zoster virus,Epstein-Barr virus EBV, Human cytomegalovirus, and human herpesvirus 7),single-stranded RNA viruses, Reo- and Retroviruses (ex. Hepatitis Bvirus, Human immunodeficiency virus 1 and 2, and Human T-lymphotropicvirus 1).

Fungal infections, as referenced herein, may include a mammalianinfection by any bacterial species. Embodiments of the fungal species,as used herein, may include, but are not limited to, Fusarium oxysporum,Pneumocystis jirovecii, Aspergillus spp., Coccidioidesimmitis/posadasii, Candida albicans, Filobasidiella neoformans,Trichosporon, Encephalitozoon cuniculi, Enterocytozoon bieneusi, Mucorcircinelloides, Rhizopus oryzae, and Lichtheimia corymbifera.

Parasite infections, as referenced herein, may include a mammalianinfection by any parasite. Embodiments of the parasite may be, but arenot limited to, Entamoeba histolytica, Babesia microti, Babesia sp. WA1,Trypanosoma cruzi, Taenia solium, Echinococcus granulosus, Leishmaniabraziliensis, L. donovani, L. tropica, Plasmodium falciparum, Plasmodiumvivax, Plasmodium ovale and Plasmodium malariae, Plasmodium knowlesi,Paragonimus westermani, chistosoma sp., S. mansoni, S. haematobium, S.japonicum, Strongyloides stercoralis, Toxocara canis, Toxoplasma gondii,Trichinella spiralis African trypanosomiasis, Ancylostoma,Angiostrongylus, Anisakis, Baylisascaris procyonis, Clonorchis(Opisthorchis) sinensis, Echinococcus multilocularis, Fasciola hepatica,Filariasis, Gnathostoma

Exemplary embodiments of diagnostic markers include those listed inTable III. Further exemplary embodiments for the diagnosis of type-IIdiabetes include, but are not limited to, Albumin, Alpha-1 antitrypsin(A1AT)-G Protein, Cystatin C, Cystatin A, alpha-2-macroglobulin (A2MG),Uteroglobin, Transthyretin (TTR), Annexin A1, Annexin A2, Annexin A3 andCalnexin.

Diagnostic markers directed towards cardiovascular disease, in anexemplary embodiment, may include, but are not limited to, any isoformof creatine kinase, troponin I and T, LD, Myoglobin, ALT/AST, H-FABP,and Glycogen phosphorylase B.

Diagnostic markers directed towards cancer, in addition to thosedescribed in Table III, may include, but are not limited to, Aldosereductase, Angiogenin, Annexin A1, B-cell activating factor (BAFF),B-cell lymphoma 2 (BCL2)-like 2, Beta Human chorionic gonadotropin,Ca15-3, Calcyclin, Calvasculin, Cathepsin D, Caveolin-1, Chromogranin A,Alpha-crystallin B chain (CRYAB), Endostatin, Eotaxin-2, Epithelial celladhesion molecule (EpCAM), Ezrin, fatty acid binding protein 4 (FABP4),Galectin-3, γ-glutamylcysteine ligase regulatory chain (GCLR), Gelsolin,Glucose 6-phosphate (G6P), Glycoprotein 130 (gp130), GlutathioneS-transferase Mu 1 (GSTM1), Hepsin, High-mobility group protein B1(HMGB-1), Insulin-like growth factor binding protein 1 (IGFBP-1),Insulin-like growth factor binding protein 4 (IGFBP-4), Insulin-likegrowth factor binding protein 5 (IGFBP-5), Insulin-like growth factorbinding protein 6 (IGFBP-6), LGL, latency associated peptide (LAP),macrophage stimulating protein (MSP), MHC class I polypeptide-relatedsequence A (MICA), Nucleoside diphosphate kinase B (NME2),Neuron-specific Enolase (NSE), Osteopontin, Osteoprotegerin, Pepsinogen,Peroxiredoxin, Phosphoserine aminotransferase (PSAT1), Prostate SpecificAntigen, Receptor tyrosine-protein kinase erbB-3 (ErbB3), Serpin B3,Vascular smooth muscle cell growth factor R2 (VSGF R2/KDR), Vascularendothelial growth factor R3 (VEGF R3/Flt-4), Thyroglobulin, Tyrosinekinase with immunoglobulin-like and EGF-like domains 2 (TIE-2), Tissueplasminogen activator (tPA), Transforming growth factor beta (TGF-β1),Tumor necrosis factor receptor 1 (TNF-R1), urokinase-type PlasminogenActivator (uPA), urokinase-type Plasminogen Activator Receptor (uPAR),BrcaI, BrcaII, kallikreins, e-cadherin, Hox peptide, and Engrailed-2.

Diagnostic markers directed towards a bacterial or virus may include anysurface or secreted antigen from the bacteria or virus, or a conservednucleotide sequence.

TABLE III Present in Present Relevant Serum/ in Class of Tumor MarkerCancer Plasma Saliva Molecules α-fetoprotein Heptacellular Yes YesProtein carcinoma Cancer Antigen Breast cancer Yes Yes Protein (CA15-3)Cancer Antigen Ovarian cancer Yes Yes Protein (CA 125) CarcinoembryonicMany epithelial Yes Yes antigen cancers Prostate specific Prostatecancer Yes Yes Protein antigen c-erb-2 Breast cancer Yes Nucleic acidP16, p53 Oral SCC BRACA1, Breast cancer Yes Yes Nucleic acid BRACA2Hemoglobin-A1c Yes Protein

B. Diagnostic Markers

Further, diagnostic markers according to an embodiment of the presentdisclosure may include one or more of: 1,25 dihydroxy-vitamin D,17-Hydroxyprogesterone, 25-hydroxy-vitamin D, Antineutrophil CytoplasmicAntibodies, 5-Hydroxy Tryptamine, 5-hydroxyindoleacetic acid,Acetoacetate, Activated Partial Thromboplastin Time, AdrenocorticotropicHormone, Alanine aminotransferase, Alanine transaminase, Albumin,Albumin-to-Creatinine ratio, Albumin/Globulin ratio, Alcohol, Aldolase,Aldosterone, Aldosterone and plasma renin activity, Aldosterone andRenin, Alkaline Phosphatase, Allergen-specific IgE, Alpha tryptase,Alpha-1 Antitrypsin, Alpha-fetoprotein, Alpha1-antitrypsin, Alzheimerbiomarkers (including Tau protein and Amyloid Beta 42 peptides),Amylase, ANCA Antibodies, ANCA/MPO/PR3Antibodies, Angiotensin-ConvertingEnzyme, Anti-citrulline antibody, anti-cyclic citrullinated peptideantibody, Anti-retroviral drug resistance testing,anti-ribonucleoprotein, anti-Sjögren's Syndrome A, anti-Sjögren'sSyndrome B, Anti-Smooth Muscle Antibody, anti-topoisomerase,Anticardiolipin Antibodies, Antidiuretic Hormone, Antifactor Xa heparin,Antiglobulin, Antihistidyl Transfer RNA, Synthase Antibodies,Antimicrosomal antibody, Antimitochondrial Antibody andAntimitochondrial M2 Antibody, Antineutrophil Cytoplasmic Antibodies,Antinuclear Antibody test, Antiphospholipid antibodies,Antiphospholipids, Antistreptolysin O titer, Antithrombin,Antithyroglobulin antibody, Antithyroid antibodies, Apolipoprotein A-I,Apolipoprotein B-100, Arginine Vasopressin, Aspartate aminotransferase,Aspartate transaminase, B-type natriuretic peptide, Beta hCG, Betatryptase, Beta-2 Microglobulin, Beta-hydroxybutyrate,Beta-hydroxybutyric acid, Beta2 Microglobulin, Bicarbonate, Bilirubin,cholesterol, Blood clotting factors, Bordetella pertussis Antibodies,Borrelia burgdorferi antibodies, IgM/IgG, Brain natriuretic peptide,Breast Cancer Gene 1 and Breast Cancer Gene 2, c-ANCA, c-erbB-2,C-peptide, C-Reactive Protein, Caffeine, Calcidiol, Calcifidiol,Calcitonin, Calcitriol, Calcium, Calcofluor white stain, Cancer Antigen125, Cancer Antigen 15-3, Cancer Antigen 19-9, Cancer antigen-breast,Cancer antigen-GI,

Carbamazepine, Carcinoembryonic Antigen, Cardiac-specific Troponin I andTroponin T, Cardiolipin Antibodies, Catecholamines, Celiac DiseaseTests, Ceruloplasmin, Chickenpox, Chickenpox and Shingles Tests,Chlamydia, Chloride, Cholesterol, Chromogranin A, Chymotrypsin,Citrulline antibody, Coagulation Factors, Cobalamin, ComplementComponent C3, Complement Component C4, Complexed PSA, Conjugatedbilirubin, Copper, Corticotropin, Cortisol, Cotinine, Creatine Kinase,Creatine Kinase-MB, Creatinine, Cryoglobulin, Cryoprotein, CyclicCitrullinated Peptide Antibody, Cyclosporine, Cystatin C, CysticFibrosis Gene Mutation Panel, Cystic fibrosis genotyping,Cytomegalovirus, Cytotoxic T-cells, Dehydroepiandrosterone Sulfate,Delta-aminolevulinic acid, Depakene, Depakote, Des-gamma-carboxyprothrombin, DHEA Sulfate, Diabetes mellitus autoantibody panel,Digoxin, Dilantin, Direct Anti-human Globulin test, Direct AntiglobulinTest, Direct bilirubin, Direct Low-density lipoprotein cholesterol,Dopamine, Drug screen, eGFR, Epidermal Growth Factor Receptor,Epinephrine, Epstein-Barr Virus Antibodies, Erythropoietin, Estradiol,Estriol, Estrogens, Estrone, Ethanol, F-Actin Antibody, Factor I, FactorV Leiden, Factor V Leiden Mutation and PT 20210 Mutation, Factor VLeiden mutation: Activated protein C resistance, Factor V R506Q,Ferritin, Fetal fibronectin, Fibrin degradation fragment, Fibrinogen,Fluorescent Antinuclear Antibody, Fluorescent treponemal antibodyabsorption, Folic Acid, Follicle-stimulating hormone, Fragment D-dimer,Fructosamine, Gamma-glutamyl transferase, Gamma-glutamyl transpeptidase,Gastrin, Genital Human Papillomavirus, Glucose-6-PhosphateDehydrogenase, Glutamic Acid Decarboxylase Autoantibodies,Gluten-Sensitive Enteropathy Tests, Glycated Albumin, Glycatedhemoglobin, Heavy Metals (such as lead, mercury, iron, copper, andzinc), Hemoglobin, Hemoglobin-binding Protein, Hemogram, HeparinAnti-Xa, Hepatitis A, Hepatitis B, Hepatitis C, Herpes Simplex Virus,Typel and Type 2, Herpes Zoster, Heterophile Antibodies, High-densitylipoprotein cholesterol, High-sensitivity C-reactive protein,Homocysteine, Human calcitonin, Human chorionic gonadotropin, Humanepidermal growth factor receptor, Human Growth Hormone, Humanimmunodeficiency virus antibody test, Human Immunodeficiency VirusGenotypic Resistance Testing, Human Leukocyte Antigen, Immunoreactivetrypsinogen, Insulin Autoantibodies, Insulin C-peptide, Insulin-likeGrowth Factor-1, Insulinoma-Associated-2 Autoantibodies, Intact PTH,Interstitial Cell Stimulating Hormone, Iron, Ischemia-Modified Albumin,Islet autoantibodies Islet Cell Cytoplasmic Autoantibodies, Janus Kinase2, Ketone bodies Ketones, blood, Lactate, Lactate dehydrogenase, LacticAcid, Lactic dehydrogenase, Lead, Lipoprotein, Lithium, Low-densitylipoprotein cholesterol, Lupus Antibody, Luteinizing hormone, Lymeantibodies IgM/IgG by Western blot, Magnesium, Mast cell tryptase,Measles or Mumps IgM and IgG Antibodies, Mercury, Metanephrine andNormetanephrine, Methotrexate, Methylmalonic Acid, Microalbumin,Mitochondrial Antibody, Mononuclear heterophile test, Mycophenolic acid,Mycoplasma, Mycoplasma pneumoniae IgG and IgM antibodies,Myeloperoxidase Antibodies, Myoglobin, N-terminal pro b-type natriureticpeptide, Neonatal bilirubin, Nicotine, Norepinephrine, p24 antigen,Parathyroid Hormone, Parvovirus, Pertussis, Phenobarbital, Phenyloin,Phosphate, Phosphorus, Platelet-activating factor acetylhydrolase,porphobilinogen, Potassium, Prealbumin, Presenilin 1 gene,Procalcitonin, Progesterone, Proinsulin C-peptide, Prolactin, ProstateSpecific Antigen, Protein C, Protein C and Protein S, Protein TyrosinePhosphatase-like Autoantibodies, Proteinase 3 Antibodies, Prothrombin20210 mutation, Prothrombin 20210 mutation: PT G20210A, factor II 20210,Red Blood Cell Antibody Identification, Renin, Respiratory SyncytialVirus, Rheumatoid Factor, Ristocetin Cofactor, Rubella, Sclerodermaantibodies, Serine Protease 3, Serotonin, Siderophilin, Sirolimus, Smithantibody, Smooth Muscle Antibody, Sodium, Soluble Mesothelin-RelatedPeptides, Somatomedin C, Somatotropin, Syphilis, Tacrolimus, Tegretol®,Testosterone, Testosterone-estrogen Binding Globulin, Theophylline,Theophylline and Caffeine, Thiopurine methyltransferase, ThiopurineS-methyltransferase, Thymotaxin, Thyrocalcitonin, Thyroglobulin, Thyroidperoxidase antibody, Thyroid stimulating hormone receptor antibody,Thyroid stimulating immunoglobulin, Thyroid-stimulating hormone,Thyroperoxidase antibody, Thyrotropin, Thyroxine, Thyroxine-bindingprealbumin, Transferrin, Transthyretin test, Treponema pallidum particleagglutination assay, Trichomonas, Trichomoniasis, Triglycerides,Triiodothyronine, Troponins, Trypsin, Trypsin and Chymotrypsin,Trypsinogen, Tryptase, Valproic acid, Vancomycin, Vanillylmandelic acid,Zoster Virus, Vasopressin, Very Low-Density Lipoprotein Cholesterol,Viral Hepatitis A Antibody, Viral Hepatitis C. Viral Load (HIV), VitaminB12, Vitamin B12 & Folate, Vitamin D, Vitamin D2, Vitamin D3, Vitamin K,von Willebrand Factor, West Nile Virus, Westergren sedimentation rate,and Zinc Protoporphyrin.

C. Exemplary Targets for Stabilizing Agent(s)

1. Amylase

Amylase is an enzyme that breaks down starch into sugar, and is found insuch places as human saliva, where it begins the chemical process ofdigestion. The α-amylases are calciummetalloenzymes that are completelyunable to function in the absence of calcium. Through acting at randomlocations on the starch chain, α-amylase breaks down carbohydrates intomaltotriose and maltose from amylase. In animals, α-amylase is a majordigestive enzyme that has an optimum pH of 6.7 to 7.0.

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may be an inhibitor of α-amylase.Further, the stabilizing agent may be active in a biological fluid, suchas in saliva. In an exemplary embodiment, a stabilizing agent maycomprise one or more of Fixanal® Buffer 6.0 (Sigma-Aldrich Co.),aluminum sulfate hydrate, aluminum hydroxide, bentonite, and aluminumpotassium sulfate dodecahydrate.

2. Lysozyme

Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase,is a glycoside hydrolase, that damage bacterial cell walls by catalyzinghydrolysis of 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins. Lysozyme is abundantin a number of secretions, such as tears, saliva, human milk, and mucus.It is also present in cytoplasmic granules of the polymorphonuclearneutrophils (PMN).

In at least one embodiment, a stabilizing agent of the presentdisclosure may be an inhibitor of lysozyme. Further, the stabilizingagent may be active in a biological fluid, such as saliva. In anexemplary embodiment, a stabilizing agent may comprise one or more ofFixanal®Buffer 6.0 (Sigma-Aldrich Co.), bentonite, benzoic acid, andacetic acid.

3. Peroxidase

Peroxidase are a class of oxidoreductase enzymes that catalyze theoxidation of a compound by the decomposition of hydrogen peroxide or anorganic peroxide. These peroxidases may be found in many differentbodily fluids. For example, saliva contains both salivary peroxidase(SPX) and myeloperoxidase (MPO). These peroxides may act on a number ofdifferent diagnostic markers found in bodily fluids to mask thedetection or analysis of the markers.

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may be an inhibitor peroxidaseactivity. Further, the stabilizing agent may be active in a biologicalfluid, such as saliva, blood, serum, or cancer cells. In an exemplaryembodiment, a stabilizing agent may comprise one or more of aluminumsulfate hydrate, benzoic acid, and aluminum potassium sulfatedodecahydrate.

4. Insulin Resistance

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may preserve a diagnostic marker forinsulin resistance and/or glucose intolerance. Further, the stabilizingagent may be active in a biological fluid, such as saliva. In anexemplary embodiment, a diagnostic marker may be one or more ofα-hydroxybutyrate, 1-linoleoyl-GPC, palmitate, Glycine,3-methyl-2-oxybutyrate. Moreover, a diagnostic marker may be any markerof early stage insulin resistance and glucose intolerance in anon-diabetic individual.

5. Galactose Oxidase

Galactose oxidase is a member of the family of oxidoreductaases, and hasbeen shown to participate in galactose metabolism. Because of thisactivity, galactose oxidase may be used to detect mucin-likeglycoproteins. These glycoproteins are a major component of mucussecreted by epithelial and glandular cells and are primarily responsiblefor the protective properties of the viscoeleastic mucous barrier.Mucins have been implicated in the process of cholesterol gallstoneformation, and has been identified as having abnormal expression in somecancers.

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may be an inhibitor of galactoseoxidase. Further, the stabilizing agent may be active in a biologicalfluid, such as saliva. In an exemplary embodiment, a stabilizing agentmay comprise one or more of aluminum potassium sulfate dodecahydrate,3-tert-butyl-hydroxyanisole, benzoic acid, and acetic acid.

6. Glycated Hemoglobin

Glycated hemoglobin (HbA1c) is a form of hemoglobin that is indicativeof the average plasma glucose concentration over a period of time. Dueto the transport of components of the plasma into bodily fluids, such assaliva, the level of HbA1c may be determined in bodily fluids inaddition to plasma.

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may be an inhibitor of HbA1cdegradation. Further, the stabilizing agent may be active in abiological fluid, such as saliva. In an exemplary embodiment, astabilizing agent may comprise one or more of acetic acid, aluminumhydroxide bentonite, aluminum sulfate hydrate, aluminum potassiumsulfate dodecahydrate, benzoic acid, caffeine, and3-tert-butyl-hydroxyanisole, or a combination thereof

7. Cancer Antigen 19-9 (CA 19-9)

Cancer Antigen 19-9 (CA 19-9), which may in some instances be referredto as Cancer Angigen 19.9, Cancer antigen-GI or CA-GI, is an antigenassociated with various cancers, such as prostate and colon cancer. Atleast one use of CA 19-9 may be see whether a pancreatic tumor issecreting antigen. If that is the case, then the levels may decreasewhen the tumor is treated, and they may rise again if the diseaserecurs.

In at least one embodiment of a stabilizing agent of the presentdisclosure, the stabilizing agent may be an inhibitor of CA 19-9degradation. Further, the stabilizing agent may be active in abiological fluid, such as saliva, blood, serum, or cancer cells. In anexemplary embodiment, a stabilizing agent may comprise one or more ofaluminum sulfate hydrate, benzoic acid, and aluminum potassium sulfatedodecahydrate.

Further, in an additional exemplary embodiment, the components of astabilizing agent may be present at or about equal amounts, such as forexample 1:1:1 in a stabilizing agent with three active components.Additionally, the stabilizing agents may in at least one embodimentinhibit degradation or inactivation of a diagnostic marker by at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 99%. Moreover, the stabilizingagent may, in at least one embodiment, inhibit degradation of adiagnostic marker for at least about 1 minute, at least about 5 minutes,at least about 10 minutes, at least about 15 minutes, at least about 30minutes, at least about 1 hour, at least about 2 hours, at least about 4hours, or at least about 8 hours.

Additional Components for Stabilizing Agent Carriers:

According to at least one embodiment of the present disclosure,stabilizing agents may be incorporated into various carriers for theiruse with mammals. Such carriers may include various rinses, gums,lozenge, mouthwash, beverages, confectionary, washes, or otherapplicable vehicles to deliver a stabilizing agent to the site of adiagnostic marker.

Embodiments of a carrier, such as a rinse, according to the presentdisclosure may also comprise one or more additional component which mayinclude an anti-caking agent, a chemical preservative, an emulsifyingagent, a nutrient and dietary supplement, a sequestrant, a stabilizer,an additive, a synthetic and flavoring substance. Exemplary embodimentsof these components are listed herein (See section entitled “AdditionalComponents for Rinse”). Further, the one or more additional componentmay be a substance which has been labeled as Generally Recognized AsSafe (GRAS) by the Food and Drug Administration of the United States ofAmerica.

At least one embodiment of a carrier of the present application maycomprise a liquid. The liquid, in at least one embodiment, is anon-toxic liquid. Further, the liquid may comprise water, a beveragecontaining water, or glycerin. Moreover, in at least one embodiment, astabilizing agent may be at least partially dissolved in the liquid.

It will be appreciated that the above list of excipients and/oradditives is provided merely by way of example and that various othersuch components may be used in the formulation of the present invention.

The following components, in at least one embodiment of the rinse of thepresent disclosure, may include:

1. ANTI-CAKING AGENTS: aluminum calcium silicate, calcium silicate,magnesium silicate, sodium calcium aluminosilicate, and tricalciumsilicate.

2. CHEMICAL PRESERVATIVES: ascorbic acid, ascorbyl palmitate, benzoicacid, butylated hydroxyanisole, butylated hydroxytoluene, calciumascorbate, calcium propionate, calcium sorbate, caprylic acid, dilaurylthiodipropionate, erythorbic acid, gum guaiac, methylparaben, potassiumbisulfite, potassium metabisulfite, potassium sorbate, propionic acid,propyl gallate, propylparaben, sodium ascorbate, sodium benzoate, sodiumbisulfite, sodium metahisulfite, sodium propionate, sodium sorbate,sodium sulfite, sorbic acid, stannous chloride, sulfur dioxide,thiodipropionic acid, tocopherols.

3. EMULSIFYING AGENTS: cholic acid, desoxycholic acid, diacetyl tartaricacid esters of (M)mono- and diglycerides, glycocholic acid, mono- anddiglycerides, monosodium phosphate derivatives of above, propyleneglycol, ox bile extract, taurocholic acid.

4. NUTRIENTS AND DIETARY SUPPLEMENTS: alanine, arginine, ascorbic acid,aspartic acid, biotin, calcium carbonate, calcium citrate, calciumglycerophosphate, calcium oxide, calcium pantothenate, calciumphosphate, calcium pyrophosphate, calcium sulfate, carotene, cholinebitartrate, choline chloride, copper gluconate, cuprous iodide,cysteine, cystine, ferric phosphate, ferric pyrophosphate, ferric sodiumpyrophosphate, ferrous gluconate, ferrous lactate, ferrous sulfate,glycine, histidine, inositol, iron (reduced), isoleucine, leucine,linoleic acid, lysine, magnesium oxide, magnesium phosphate, magnesiumsulfate, manganese chloride, manganese citrate, manganese gluconate,manganese glycerophosphate, manganese hypophosphite, manganese sulfate,manganous oxide, mannitol, methionine, methionine hydroxy analogue,niacin, niacinamide D-pantothenyl alcohol, phenylalanine, potassiumchloride, potassium glycerophosphate, potassium iodide, proline,pyridoxine hydrochloride, riboflavin, riboflavin-5-phosphate, serine,sodium pantothenate, sodium phosphate, sorbitol, thiamine hydrochloride,thiamine mononitrate, threonine, tocopherols, tocopherol acetate,tyrosine, valine, vitamin A, vitamin A acetate, vitamin A palmitate,vitamin B12, vitamin D2, vitamin D3, zinc sulfate, zinc gluconate, zincchloride, zinc oxide, zinc stearate.

5. SEQUESTRANTS: calcium acetate, calcium chloride, calcium citrate,calcium diacetate, calcium gluconate, calcium hexametaphosphate, calciumphosphate. monobasic, calcium phytate, citric acid, dipotassiumphosphate, disodium phosphate, isopropyl citrate, monoisopropyl citrate,potassium citrate, sodium acid phosphate, sodium citrate, sodiumdiacetate, sodium gluconate, sodium hexametaphosphate, sodiummetaphosphate, sodium phosphate, sodium potassium tartrate, sodiumpyrophosphate, sodium pyrophosphate, tetra, sodium tartrate, sodiumthiosulfate, sodium tripolyphosphate, stecaryl citrate, tartaric acid.

6. STABILIZERS: acacia (gum arabic), agar-agar, ammonium alginate,calcium alginate, carob bean gum, chondrus extract, ghatti gum, guargum, potassium alginate, sodium alginate, sterculia (or karava) gum,tragacanth.

7. ADDITIVES: acetic acid, adipic acid, aluminum ammonium sulfate,aluminum potassium sulfate aluminum sodium sulfate, aluminum sulfate,ammonium bicarbonate, ammonium carbonate, ammonium hydroxide, ammoniumphosphate, ammonium sulfate, bees wax, bentonite, butane, caffeine,calcium carbonate, calcium chloride, calcium citrate, calcium gluconate,calcium hydroxide, calcium lactate, calcium oxide, calcium phosphate,caramel, carbon dioxide, carnauba wax, citric acid, dextrans, ethylformate, glutamic acid, glutamic acid hydrochloride, glycerin, glycerylmonostearate, helium, hydrochloric acid, hydrogen peroxide, lactic acid,lecithin, magnesium carbonate, magnesium hydroxide, magnesium oxide,magnesium stearate, malic acid, methylcellulose, monoammonium glutamate,monopotassium glutamate, nitrogen, nitrous oxide, papain, phosphoricacid, potassium acid tartrate, potassium bicarbonate, potassiumcarbonate, potassium citrate, potassium hydroxide, potassium sulfate,propane, propylene glycol, rennet, silica aerogel, sodium acetate,sodium acid pyrophosphate, sodium aluminum phosphate, sodiumbicarbonate, sodium carbonate, sodium citrate, sodiumcarboxy-methylcellulose, sodium caseinate, sodium citrate, sodiumhydroxide, sodium pectinate, sodium phosphate, sodium potassiumtartrate, sodium sesquicarbonate, sodium tripolyphosphate, succinicacid, sulfuric acid, tartaric acid, triacetin, triethyl citrate.

8. SYNTHETIC FLAVORING SUBSTANCES: acetaldehyde, acetoin, aconitic acid,anethole, benzaldehyde, N-butyric acid, d- or l-carvone cinnamaldehyde,citral, decanal, diacetyl, ethyl acetate, ethyl butyrate, ethylvanillin, eugenol, geraniol, geranyl acetate, glycerol tributyratelimonene, linalool, linalyl acetate, l-malic acid, methyl anthranilate,3-methyl-3-phenyl glycidic acid, ethyl ester, piperonal, vanillin.

II. Indicator Carriers

In at least one additional embodiment of a carrier, of the presentdisclosure, the carrier comprises an indicator compound capable ofbinding to and/or reacting with a target to produce an indicator signal.The target in an exemplary embodiment may be a diagnostic marker, suchas the presence of glucose or glucose in excess of a defined threshold,the presence of one or more glycosylated protein related to diabetes orpre-diabetes, the presence of cancer (or pre-cancer) markers, and thepresence of cardiac (or pre-cardiac) markers.

A. Indicators

The indicator compound in an exemplary embodiment of a carrier accordingto the present disclosure may be any compound, chemical, or biologicalcomponent which may interact with a target or byproduct of a target. Forexample, an indicator compound may comprise an antibody, a reactivechemical compound, a labeled molecule, or any combination thereof.Antibodies used in an embodiment of the present disclosure may bemonoclonal or polyclonal and derived from any species (e.g. human, rat,mouse, rabbit, pig). Further, indicator molecules may be aptamers,proteins, peptides, small organic molecules, natural compounds (e.g.steroids), non-peptide polymers, WIC multimers (includingMHC-dextramers, MHC-tetramers, MHC-pentamers and other MHC-multimers),or any other molecules that specifically and efficiently bind to othermolecules are also marker molecules.

Labeled molecules, for use as indicator compounds, may be any moleculethat absorbs, excites, or modifies radiation, such as the absorption oflight (e.g. dyes and chromophores) and the emission of light afterexcitation (fluorescence from fluorochromes). Additionally, labeledmolecules may have an enzymatic activity, by which it catalyzes areaction between chemicals in the near environment of the labelingmolecules, producing a signal which include production of light(chemi-luminescence) or precipitation of chromophors, dyes, or aprecipitate that can be detected by an additional layer of detectionmolecules.

Fluorescence labels may produce the presence of light at a singlewavelength, or a shift in wavelengths. Exemplary fluorescent labels mayinclude:

-   -   Fluor dyes, Pacific Blue™, Pacific Orange™, Cascade Yellow™;    -   AlexaFluor® (AF); o AF405, AF488.AF500, AF514, AF532, AF546,        AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700,        AF710, AF750, AF800;    -   Fluorescein (Flu) or any derivate of that, ex. FITC (fluorescein        isothiocyanate) Cy-Dyes o Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7        Fluorescent Proteins; o RPE (R-phycoerythrin), PerCp, APC        (Allophycocyanin); other of phycobillin containing proteins,        e.g. phycobiliprotein o Green fluorescent proteins (GFP);    -   GFP and GFP derivated mutant proteins; BFP1CFP, YFP, DsRed, T1,        Dimer2, mRFP1, MBanana, mOrange, dTomato, tdTomato, mTangerine,        mStrawberry, mCherry Tandem dyes: o RPE-Cy5, RPE-Cy5.5, RPE-Cy7,        RPE-AlexaFluor® tandem conjugates;    -   RPE-Alexa610, RPE-TxRed o APC-Aleca600, APC-Alexa610, AP        C-Alexa750, APC-Cy5, APC-Cy5.5 Multi fluorochrome assemblies o        Multiple fluorochromes attached to a polymer molecule, such as a        peptide/protein, Dex, or poly-sacceride. o Any combination of        the fluorescent dyes involving in generation of FRET;    -   (Fluorescence resonance energy transfer) based techniques,        Multiple fluorochromes associated or coupled to a polymeric        molecule, or consisting of polymeric residues, lonophors; ion        chelating fluorescent probes or Probes that change wavelength        when binding a specific ion, such as calcium;    -   Probes that change intensity when binding to a specific ion,        such as calcium; and    -   Combinations of fluorochromes on the same marker. The marker is        not identified by a single fluorochrome but by a code of        identification being a specific combination of fluorochromes, as        well as inter related ratio's of intensities.

B. Target

The target of an exemplary indicator compound of the present disclosuremay be any diagnostic marker or diagnostic condition for a disease state(or pre-disease state) of an individual. For example, the disease statemay be diabetes, pre-diabetes, cardiac disease, pre-cardiac conditions,cancers of any stage and type, or pre-cancerous conditions.Specifically, the target may be the presence, or level of, glucose foundin saliva for diabetes. Alternately, the target may be the presence, orlevel of, glycosylated proteins, such as advanced glycosylation endproducts. Further, the target may be the presence or level of cardiacmarkers, such as Creatine kinase-MB (CK-MB), myoglobin, homocysteine,C-reactive protein (CRP), troponin T (cTnT), and troponin I (cTnI).Moreover, the target may be the presence of cancer markers, such asCancer Antigen 125, Cancer Antigen 15.3, Cancer Antigen 19.9, Prostatespecific antigen, Carcinoembryonic Antigen, Alpha Feto-Protein,Epidermal growth factor receptor, Kallikrein 3, Vascular endothelialgrowth factor A (VEGF), Calcitonin, Chromogranin A, Gastrin, S100 alphachain, Somatostatin, Thyroglobulin, V-erb-b2, cyckub-dependent kinaseinhibitor 1 (p21), Breast Cancer Antigen 1 and 2 (BRCA1 and 2), MutLhomolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), andpostmeiotic segregation increased 1 and 2 (PMS1 and 2). Moreover, thetarget may be the presence or level of any diagnostic marker includedherein.

C. Indicator Signal

The indictor signal in at least one embodiment of the present disclosuremay comprise any detectable signal, including but not limited to, colorchange, fluorescence, and chemical/structural change of the target(and/or indicator compound) so as to be amenable to reacting with asecondary detection marker. Further, the detection of a signal mayinvolve a secondary reactive molecule.

D. Glucose

In an embodiment of the present disclosure, glucose may be detectedthrough an indicator rinse by a chemical or enzymatic method. Exemplaryembodiments of this method may include alkaline copper reduction,alkaline ferricyanide reduction, glucose oxidase, and/or hexokinase.

In an exemplary embodiment of an indicator rinse of the presentdisclosure, the rinse may further comprise one or more additionalcomponent as described herein. These additional components may includestabilizing agents, as well as GRAS components (such as those listedherein).

E. Glycosylated Hemoglobin

In an embodiment of the present disclosure, glycated Hemoglobin A1c(HbA1c) may be detected through any known method for the detection ofHbA1c or fragments thereof, such as high pressure liquid chromatography,immunoassays, and an indicator rinse by a chemical or enzymatic method.In an exemplary embodiment of an indicator rinse of the presentdisclosure, the rinse may further comprise one or more additionalcomponent as described herein. These additional components may includestabilizing agents, as well as GRAS components (such as those listedherein).

F. Cancer Antigen 19-9

In an embodiment of the present disclosure, cancer antigen 19-9 (CA19-9) may be detected through any known method for the detection of CA19-9 or fragments thereof, such as high pressure liquid chromatography,immunoassays, and an indicator rinse by a chemical or enzymatic method.In an exemplary embodiment of an indicator rinse of the presentdisclosure, the rinse may further comprise one or more additionalcomponent as described herein. These additional components may includestabilizing agents, as well as GRAS components (such as those listedherein).

III. Diagnostic Marker Detection

In an embodiment of the present disclosure, a diagnostic markersusceptible to degradation or alteration by a destructive component maybe detected through any known method, including, but not limited to,high pressure liquid chromatography, immunoassays, and an indicatorrinse by a chemical or enzymatic method. Further, the detection may beconducted in a micro well, deep well, microarray, microfluidic device,high throughput screen, or any applicable diagnostic device.

A. Rapid Diagnostic

Detection of a diagnostic marker may be conducted according to a rapiddetection system of the present disclosure. Turning to FIG. 1A, adetection platform 100 may comprise a platform 102 having a plurality oftest wells 104, where each well is capable of containing a plurality oftest spots 106. The plurality of test wells 104 may include at least 2,at least 4, at least 8, at least 16, at least 32, at least 64, or atleast 256 wells. The plurality of tests spots 106 may include at least2, at least 4, at least 8, at least 16, at least 32, or at least 64 testspots 106. Further, each of the plurality of test spots 106 may compriseone or more diagnostic marker or control marker.

An embodiment of detection platform 100 may further be sized and shapedto allow for automated detection and analysis of a diagnostic or controlmarker. In at least one embodiment, the detection system 100 maycomprise an identifier 108, such as a barcode or Radio-frequencyidentification (RFID) tag, to allow for the identification of thedetection system 100 and data collected from the analysis of test spots106.

Further, detection platform 100 may be comprised of any applicablematerial sufficient to house or define test wells 104. Exemplaryembodiments of applicable materials for detection platform 100 may be apolymer, glass, metal, quartz, nylon, silica-based material, resin, orcombination thereof. Further, embodiments of test wells 104 may includenot only a depression defined or housed by platform 102, but may alsoinclude a defined area or region of the platform 102 (such as may befound on a microarray based platform).

Turning to FIG. 1B, at least one embodiment of detection system 150 ofthe present disclosure is shown. An exemplary embodiment of detectionsystem 150 comprises an embodiment of detection platform 100, adetection device 120 capable of determining a binding characteristicbetween a detection agent and a diagnostic marker on the detectionplatform 100, and a computer processor 130 coupled to a computerdatabase 135 and to the detection device 120. In at least embodiment,the computer processor 130 controlling the detection device 120 is ableto (1) determine the binding characteristic of a detection agent to adiagnostic agent, (2) compare the binding characteristic among each of aplurality of samples tested, (3) generate a binding report using hecompared binding characteristics, and deliver the binding report to arecipient or external computer (not shown) in communication with thecomputer processor 130.

A binding characteristic as used herein may include any measurement ofthe attraction (or repulsion) of two molecules (such as a detectionagent and a diagnostic marker).

Detection system 100 may also comprise an embodiment of a bead-basedsystem of the present disclosure having at least one bead type. Forexample, a dual bead system may be used wherein at least one bead typeis magnetic or paramagnetic. These beads may also be bound to a carrierprotein with a conjugated hapten. Further, the beads may have an analytecapture monoclonal antibody reagent bound to it. Additionally, thebeads, in at least one embodiment, do not retain their magneticproperties when removed from a magnetic field. In at least oneembodiment, the beads may be Europium micro-particles. The sizes of theEuropium micro-particles may be varied as needed to accommodate themethod of detection used.

At least one embodiment of a bead based system may comprise a latexparticle (See FIGS. 2-4). The latex particle according to at least oneembodiment may be a Carboxylate modified latex (CML) bead. Further, anexemplary embodiment of a bead may include bovine serum albumin (BSA)and/or human serum albumin (HSA). Additionally, an embodiment of a beadbased system of the present disclosure may include a linker coupled toHSA and/or BAS. The linker according to an embodiment, may comprise amaleimide compound. At least one exemplary embodiment of a maleimidecompound may be Succinimidyl 4-[N-Maleimidomethyl]Cyclohexane-1-Carboxylate (SMCC) or N-Hydroxysuccinimide-activatedhexa(ethylene glycol) undecane Thiol (NHS). Moreover, at least oneexemplary embodiment of a bead based system may further comprise anantibody linked to maleimide on the bead. Accordingly, at least oneembodiment of the bead based system comprises a latex particle coupledto BAS or HSA, maleimide coupled to BAS or HSA, and an antibody coupledto maleimide.

The latex particle, according to at least one embodiment may be in asize range from about 0.02 μm to about 7.0 μm.

Turning to FIG. 5, at least one at least one method 500 of coupling anantibody to a bead comprises the step 502 of modifying an embodiment ofa bead (such as a latex bead/particle) with BSA or HAS, wherein the BSAor HSA may further comprise a maleimide containing molecule, such asSMCC or NHS. Additionally, an embodiment of the method 500 may furthercomprise the step 504 of attaching a diagnostic marker binding agent(such as an antibody) to the bead. Step 504 of attaching the diagnosticmarker binding agent to the bead may be accomplished through binding thediagnostic marker binding agent to maleimide. Further, method 500 mayalso comprise the step 506 of attaching one or more stabilizing agent toan embodiment of a bead. Additionally, while an embodiment of method 500may use latex beads for attachment of a maleimide containing moleculeand/or a stabilizing agent, alternate embodiments of the bead maycomprise silicon, a polymer, or other applicable materials.

According to at least one embodiment of the present disclosure, one ormore stabilizing agent may be attached or adsorbed to any one of anembodiment of a detection platform, test strip, microchip, ormicrofluidic device.

B. Test Strip

Turning to FIGS. 6A and B, exemplary embodiments of a diagnostic testingdevice 600 are described. An exemplary embodiment of diagnostic testingdevice 600 comprises housing structure 602 which comprises a collectionchamber 604 capable of receiving body fluid, at least one membrane strip606 in fluid communication with collection chamber 604, animmunoassay-based fingerprint acquisition pad 608 in fluid communicationwith the collection chamber 604, and a plurality of reaction zones 610which may allow the visual display of the presence of a predetermineddiagnostic marker. For example, reaction zone 610 may in an exemplaryembodiment be capable of displaying a characteristic of thepredetermined diagnostic marker, such as the presence of, theconcentration of, or a concentration above or below a predeterminedvalue for the predetermined diagnostic agent.

In an exemplary embodiment of diagnostic testing device 600, diagnostictesting device 600 further comprises a fluid collector 612 which isoperable to collect body fluid from a subject, and wherein the fluidcollector is capable of supply collected body fluid to collectionchamber 604.

Exemplary embodiments of test strip 606 may comprise any materialcapable of adsorbing or attaching a stabilizing agent and may bind adiagnostic marker. Further, exemplary embodiments of test strip 606 maycomprise compositions comprising a polyvinyl chloride-silicacombination, nitrocellulose, or any suitable synthetic, resinousmaterial. At least one embodiment, housing 602 is capable of reducingthe exposure of the test strip 606 at sites other than the collectionchamber 604 to foreign material. Test strip 606 in at least oneembodiment may be capable of interfacing with an analysis device 615.The analysis device 615 in an exemplary embodiment may be capable ofmeasuring the level of the predetermined diagnostic marker in thesample.

Turning to FIG. 6C, an exemplary embodiment of a microarray system 620is described. An exemplary embodiment of microarray system 620 maycomprise a microarray product 622 having a microarray identifier 624,and a plurality of microarray sites 626, a control microarray product628 located on at least one microarray site 626 and bound to microarrayproduct 622, and operably connected to a computer processor 130 forproviding information regarding the identification and concentration ofmarkers on the microarray product 622 based on the microarray identifier624. Additionally, microarray product 622 may further comprise, in atleast one embodiment, an embodiment of a stabilization agent 630 locatedon at least one microarray site 626 and bound to microarray product 622.Further, microarray product 622 may comprise one or more diagnosticmarker 632 located on at least one microarray site 626 and bound tomicroarray product 622. Moreover, microarray system 620, in at least oneembodiment, may further comprise a microarray detector 634 operable todetect a signal from at least one of the control microarray product 628and/or diagnostic marker 632.

A microchip or microarrays may refer to an array of distinctpolynucleotides affixed to a substrate, such as glass, plastic, paper,nylon or other type of membrane, filter, chip, or any other suitablesolid support. The polynucleotides can be synthesized directly on thesubstrate, or synthesized separate from the substrate and then affixedto the substrate. In an embodiment, the microarray may be prepared andused at least according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

Turning to FIG. 6D, an exemplary embodiment of microfluidic device 640is described. An embodiment of microfluidic device 640 comprises asample reservoir 642 for receiving a body fluid (or other fluid sample)through a sample input 644, where the sample reservoir 642 is fluidlyconnected to a detector array 646. Additionally, in at least oneembodiment of microfluidic device 640, detector array 646 comprises areaction site 647 and a results display 648. Moreover, an exemplaryembodiment of microfluidic device 640 may further comprise a filterdevice 650 capable of separating at least one unwanted component from afluid sample and fluidly coupled between sample reservoir 642 anddetector array 646. Detector array 646 may also, in at least oneembodiment comprise a control reagent display 652, where at least onecontrol reagent may be visually detected.

Further, microfluidic device 640 may also, in at least one embodiment,be able to couple to a processor 655. Processor 655 may be able tocompare at least one reading, such as from results display 648 orcontrol reagent display 652 from an embodiment of microfluidic device640 to at least one additional stored reading on a computer database 657of the processor 655. An embodiment of microfluidic system 660 of thepresent disclosure, may comprise one or more microfluidic device 640operationally coupled to a processor 655.

Embodiments of microfluidic devices 640, which may also be referred toas “lab-on-a-chip” systems, biomedical micro-electro-mechanical systems(bioMEMs), or multicomponent integrated systems, are exemplarykits/systems for analyzing polynucleotide regions. Such systems mayminiaturize and compartmentalize processes such as probe/targethybridization, nucleic acid amplification, and capillary electrophoresisreactions in a single functional device. Such microfluidic devicestypically utilize detection reagents in at least one aspect of thesystem, and such detection reagents may be used to detect one or morepolynucleotide region of the present disclosure. Exemplary microfluidicdevices 640 may comprise a pattern of microchannels designed onto aglass, silicon, quartz, or plastic wafer included on a microchip. Themovements of the samples may be controlled by electric, electroosmoticor hydrostatic forces applied across different areas of the microchip tocreate functional microscopic valves and pumps with no moving parts.Varying the voltage can be used as a means to control the liquid flow atintersections between the micro-machined channels and to change theliquid flow rate for pumping across different sections of the microchip.

In at least one embodiment of a microarray or microfludic device 640,the microarray or microfluidic device may comprise one or morestabilizing agent capable of reducing the degradation of a predefineddiagnostic marker. For example, a stabilizing agent may be incorporatedonto the surface of the microarray device, and/or on at least one partof the microfluidic device.

IV. Methods of Biomarker Stabilization

In at least one embodiment of a method for stabilizing biomarkers, asdescribed in FIG. 7, the method 700 comprises the step 702 of mixing abody fluid with an embodiment of a stabilizing agent. Optionally, thestep of mixing a body fluid with a stabilizing agent may comprise thestep 704 of introducing the stabilizing agent into the patient, such asthrough the oral cavity, so that the stabilizing agent mixes with thebodily fluid, and the step 706 of retrieving a body fluid comprising astabilized diagnostic marker. In an additional exemplary embodiment of amethod of stabilizing a diagnostic marker, the method comprises the step708 of isolating a body fluid having a diagnostic marker, the step 710of treating the body fluid with an embodiment of a carrier, such as arinse, gum, or beverage, as described herein, and the step 712 ofanalyzing the diagnostic marker. Isolation of the body fluid may occurthrough any customary mechanism, including, but not limited to, rinsing,swabbing, suction, collection, and lavage. Further, the rinse in anexemplary embodiment of a method 700 of the present disclosure, may beingested to stabilize a diagnostic marker present in the bodily fluid,such as in urine, semen, anal secretions, and vaginal secretions.

The step 702 of mixing a body fluid with a carrier may occur prior to,or after isolation step 708 of the body fluid. As an exemplaryembodiment, a rinse may be used to rinse the mouth. Afterwards, therinse containing the treated body fluid may be collected by spitting,suction, or other means. Alternately, expirated saliva may be mixed witha rinse to treat the saliva ex vivo. Similar treatment of body fluidsmay be performed with any type of body fluid.

In analyzing the treated body fluid, the diagnostic marker may beanalyzed through any known means. Optionally, the analysis of thetreated body fluid may include the separation of solid materials fromsoluble materials through means such as filtration, or bycentrifugation. Analysis of the treated body fluid may use anyappropriate technique, such as western blot analysis, Enzyme-LinkedImmunosorbent Assay (ELISA), protein activity assays, reversetranscription polymerase chain reaction (RT-PCR), microarray, highpressure liquid chromatography, or any comparable assay to determine acharacteristic of the diagnostic marker. Such an analysis, in anexemplary embodiment, may be of a modified product, a cleavage product,or cleavage pattern, of a diagnostic marker.

In an exemplary embodiment of the analyzing step 712, the treated bodyfluid may be placed in contact with an embodiment of a test strip, suchas a nitrocellulose strip, prior to detection with an appropriate probespecific for the diagnostic marker. The nitrocellulose strip may bedesigned to trap or filter particulates in the body fluid. This mayreduce or eliminate potential contaminants or other substances in theoral fluid that would otherwise reduce the signal to noise ration duringthe detection step. A probe (such as an antibody) with affinity to thediagnostic marker may be affixed or immobilized to a specific locationon the nitrocellulose membrane for detection of the analyte. Utilizingstandard principles of immunoassay detection (or nucleotide detection),the signal generated may be visible to the eye or detectable by aninstrument.

In an exemplary embodiment of a method of analyzing a stabilizeddiagnostic marker of the present disclosure as depicted in FIG. 8, themethod 800 comprises the step 802 of operating a system for thedetection of a diagnostic marker in a body fluid, where the systemcomprises a diagnostic device having a plurality of test wells eachcapable of containing at least one diagnostic marker binding agent, anda detection device capable of interacting with diagnostic device,wherein the detection device is capable of detecting an interactionbetween the at least one diagnostic marker binding agent and adiagnostic marker. Following step 802, the sample of bodily fluid may bediluted in step 803 with a reagent which may contain an embodiment ofstabilizing agent as described herein. At least one embodiment of method800 further comprises the steps of: contacting 804 a sample of a bodilyfluid with at least one diagnostic marker binding agent, incubating 806the contacted sample in the test well at a first temperature for apre-determined period of time. The step 804 of contacting of the sampleof bodily fluid with at least one diagnostic marker binding agent may beaccomplished through a magnetic field applied to the test well.

In at least one embodiment of method 800, the first temperature may beat room or at body temperature (such as about 22° C. or about 37° C.).The pre-determined period of time may, for example, be between five andfifteen minutes. The incubated and contacted sample may then be removedin step 808 from the plurality of test wells, and the test wellsanalyzed in step 810 to detect a binding event with the detectiondevice. Alternately, a stabilizing agent may be incubated with thediagnostic marker binding agent (also referred to as a detection agent)prior to introduction of a bodily fluid. In at least one embodiment ofthe method, each diagnostic marker binding agent may have a differentmonoclonal antibody designed to bind a specific hapten molecule.

Following the detection of a characteristic of a treated diagnosticmarker (the results of which may be termed a “profile” such as a “cancerprofile”), the characteristic may in step 812 be compared to a standardvalue to determine the presence or absence of a disease state. Thecharacteristic determined may be an activity level, the concentration ofthe diagnostic marker, or a particular modification, such asglycosylation or methylation.

Optionally, an embodiment of the method 800 may further comprise thestep 814 of customizing a detection method through screening ofapplicable pre-clinical samples through a database of stabilizingagents/cocktails. Further, an embodiment of method 800 of diagnosticmarker detection may further comprise the step 816 of comparing a levelor characteristic (such as a predictable degradation products) of adiagnostic agent with a library of known characteristics. Moreover, anexemplary embodiment of method 800 may additionally comprise the step818 of determining a diagnosis of a disease state using the comparedcharacteristic. In at least one embodiment, a method of diagnosticmarker detection may be automated, and capable of detecting at leastabout ten thousand samples in a twenty-four hour period. Method 800 inat least one embodiment, may be at least partially completed by acomputer processor. Additionally, the processor may be in communicationwith at least one more processor and/or a computer database. Forinstance, in at least one embodiment of method 800, most of the steps ofthe method may be completed with or monitored by a computer processor.Moreover, method 800, in at least one embodiment, may use a computerprocessor to perform additional step 820 of generating a report by usinga comparison of determined characteristics (such as a binding reportwhere a comparison is performed of binding characteristics), and step822 of delivering the report to a recipient (such as a user, orsecondary processor).

A method of biomarker stabilization may, in an exemplary embodiment, maybe used to diagnose a particular disease state or condition of apatient. For example, a disease state or condition as used with thismethod may include one or more of Acid-Base Disorders, Acidosis andAlkalosis, Acidosis/Alkalosis, Acute inflammatory demyelinatingpolyneuropathy, Acute myocardial infarct, Addison's Disease, AdrenalInsufficiency, Adrenal Insufficiency & Addison's Disease, Alcoholdependence, Alcoholism, Allergies, Alzheimer's Disease, Anemia, AnginaPectoris, Anthrax, Arthritis, Asthma, Atypical Pneumonia, AutoimmuneDisorders, Autoimmune thyroiditis, Avian flu, Benign ProstaticHyperplasia, Benign Prostatic Hypertrophy, Bioterrorism Agents, BleedingDisorders, Bone Marrow Disorders, Breast Cancer, Cardiovascular Disease,Celiac Disease, Cervical Cancer, Chlamydia, Chronic Fatigue and ImmuneDysfunction Syndrome, Chronic Fatigue Syndrome, Chronic thyroiditis,Colon Cancer, Community-Acquired Pneumonia, Congestive Heart Failure,Conn's Syndrome, Cushing's Syndrome, Cystic Fibrosis, Degenerative JointDisease, Diabetes, Diarrhea, Diffuse thyrotoxic goiter, Diseases of thePancreas, Disseminated lupus erythematosus, Double pneumonia, DownSyndrome, Encephalitis, Endocrine Syndromes, Endocrine System andSyndromes, Epilepsy, Fibromyalgia, Flu, Folate Deficiency, FungalInfections, Genital Herpes, Gonorrhea, Gout, Gouty arthritis, Graves'Disease, Guillain-Barre Syndrome, Influenza H1N1, Hashimoto'sThyroiditis, Healthcare-Associated Pneumonia, Heart Attack, Heart Attackand Acute Coronary Syndrome, Heart Disease, Hemochromatosis, Hepatitis,Herpes, Herpes Zoster, High blood pressure, Hospital-Acquired Pneumonia,Human Immunodeficiency Virus, Human Papillomavirus, HypercoagulableDisorders, Hypersensitivity, Hypertension, Hyperthyroidism,Hypothyroidism, Infectious Arthritis, Infectious polyneuritis,Infertility, Inflammatory Bowel Disease, Influenza, Influenza A,Influenza B, Insulin Resistance, Jaundice, Juvenile RheumatoidArthritis, Kidney and Urinary Tract Function, Disorders, and Diseases,Kidney Disease, Landry's ascending paralysis, Lead Poisoning, Leukemia,Liver Disease, Lobar pneumonia, Lower Respiratory Tract Infection, LungDiseases, Lupus, Lupus erythematosus, Lyme Disease, Lymphoma,Malnutrition, Meningitis, Meningitis and Encephalitis, Menopause,Metabolic Syndrome, Metabolic Syndrome/Syndrome X, Multiple Myeloma,Multiple Sclerosis, Myeloproliferative Disorders, Myocardial infarct,Neural Tube Defects, Nontuberculous Mycobacteria, Osteoarthritis,Osteoporosis, Ovarian Cancer, Pancreatic Cancer, Pancreatic Diseases,Pancreatic Insufficiency, Pancreatitis, Pelvic Inflammatory Disease,Peptic Ulcer, Pituitary Disorders, Pneumonia, Polycystic ovariansyndrome, Pregnancy, Primary hyperaldosteronism, Prostate Cancer,Proteinuria, Rheumatoid Arthritis, Septic Arthritis, SexuallyTransmitted Diseases, Sexually transmitted infections, Shingles, SickleCell Anemia, Sickle Cell Disease, Sjögren's Syndrome, Staph WoundInfections, Staph Wound Infections and Methicillin ResistantStaphylococcus aureus, Stein-Leventhal syndrome, Stroke, Swine flu,Syphilis, Systemic Lupus Erythematosus, Testicular Cancer, Thalassemia,Thyroid Diseases, Travelers' Diseases, Trichomonas, Tuberculosis, Typesof Liver Disease, Urinary Tract Infection, Venereal diseases, Vitamin B12 and Folate Deficiency, Vitamin B 12 Deficiency, Vitamin K Deficiency,Walking pneumonia, West Nile Virus, Wilson's Disease, Wound and SkinInfections.

V. Methods to Determine Efficacy and Specificity of a Therapeutic

According to FIG. 9, in at least one embodiment of the presentdisclosure, a method 900 of improving biomarker availability isdescribed and shown. Method 900 comprises the step 902 of contacting adiagnostic marker from a patient with a stabilization agent, the step904 of introducing a therapeutic agent to the diagnostic marker and thestabilization agent, and the step 906 of analyzing the interaction ofthe therapeutic agent with the diagnostic marker to determine atherapeutic profile, such as the binding affinity of the agent to thediagnostic marker. Further, method 900 may also comprise the step 908 ofcomparing the therapeutic profile with at least one other therapeuticprofile to determine a stabilizing impact from the stabilizing agent onthe diagnostic marker. Step 908 of comparing a plurality of therapeuticprofiles may be performed using a computer processor and may furtherutilize a computer database of previously documented therapeuticprofiles. Moreover, in an exemplary embodiment of method 900, the step902 of contacting a diagnostic marker with a stabilization agent may berepeated with a plurality of different stabilization agents and/ortherapeutic agents. Additionally, the therapeutic profile may be used todetermine a therapeutic course to treat the patient.

In FIG. 10, a method of identifying diagnostic markers of a diseasestate is shown. Method 1000, according to at least one embodiment,comprises the step 1010 of contacting a bodily fluid from a subjecthaving a disease state with a stabilizing agent, and the step 1020 ofanalyzing the stabilized bodily fluid to determine at least one propertyof a diagnostic agent, to create a disease state profile. An exemplaryembodiment of method 1000 may further comprise the step 1030 ofcomparing the disease state profile with a profile generated in a likemanner from a subject not having the disease state, and the step 1040 ofgenerating a disease state identifier panel from the compared diseasestate profile. In at least one embodiment of method 1000, the diseasestate is cancer. Exemplary embodiments of cancer may include at leastbladder cancer, lung cancer, breast cancer, melanoma, colon and rectalcancer, non-hodgkin lymphoma, endometrial cancer, pancreatic cancer,kidney (renal cell) cancer, prostate cancer, leukemia, and thyroidcancer. In at least one embodiment of method 1000, the subject iscurrently receiving treatment with an original treatment agent, and thedisease state profile may be compared with a disease profile of the samesubject while receiving a different treatment agent, a differentconcentration of the same treatment agent, or at an earlier point oftime.

In FIG. 11, an exemplary embodiment of a method for screening biomarkersis described. Method 1100 comprises the steps of (a) contacting 1110 acell from a subject having a disease state with a stabilizing agent, (b)incubating 1120 the stabilized cell with a therapeutic compound, and (c)analyzing 1130 the incubated cell for at least one diagnostic marker tocreate a marker profile. Optionally, method 1100 may further comprisethe step 1140 of comparing the marker profile with at least oneadditional marker profile to determine the efficacy of the therapeuticcompound.

In at least one method of stabilizing diagnostic markers of the presentdisclosure, as shown in FIG. 12, the method 1200 comprises the step 1210of measuring the level of diagnostic marker in a subject with a diseasestate, the step 1220 of treating the subject with a therapeutic agent,and the step 1230 of determining the post-treatment level of diagnosticmarker. Wherein the step 1210 of measuring the level of diagnosticmarker includes the step of incubation of the diagnostic marker with astabilization agent.

In at least one embodiment of method 900, 1000, 1100, or 1200, themethod may be used with a device as described herein, such as anembodiment of diagnostic system 100, test strip 600, a microarray 640,or microfluidic device 660, any of which may also be performed inconjunction with a computer processor.

VI. Computational Analysis

In at least one embodiment of the systems, methods, and devices of thepresent disclosure, a computer processor and a database may be used inor during the system, method, or device. An exemplary embodiment of asystem framework comprising at least one computer processor and at leastone database, as may be used in the embodiments of the presentdisclosure is shown in FIG. 13.

In at least one embodiment, a result from a diagnostic method may becompared using a computer processor to at least one additional resultfrom the diagnostic test, and/or to a result stored on a database. Sucha comparison, in at least one embodiment of the present disclosure, mayreveal the stabilizing agent with a greater effect on the bindingcharacteristic, may allow for the diagnosis of a disease state orhealth/lifestyle characteristic. Further, such a comparison, in at leastone embodiment, may be used to develop, confirm, or modify a therapeutictreatment of a patient having a disease state or health/lifestylecharacteristic.

As shown in exemplary system framework 1300 shown in FIG. 13, one ormore user computers 1302 may be operably connected to a system server1304. A user computer 1302 may be a computer, computing device, orsystem of a type known in the art, such as a personal computer,mainframe computer, workstation, notebook computer, laptop computer,hand-held computer, wireless mobile telephone, personal digitalassistant device, and the like.

One or more administrator computers 1306 may also be operably connectedto system server 1304 including through a network 1308 such as theInternet. Administrator computers 1306, similar to user computers, maybe computers, computing devices, or systems of a type known in the art,such as personal computers, mainframe computers, workstations, notebookcomputers, laptop computers, hand-held computers, wireless mobiletelephones, personal digital assistant devices, and the like. Inaddition, user computers and administrator computers may each comprisesuch software (operational and application), hardware, and componentryas would occur to one of skill of the art, such as, for example, one ormore microprocessors, memory, input/output devices, device controllers,and the like. User computers and administrator computers may alsocomprise one or more data entry means (not shown in FIG. 13) operable bya user of client computer and/or an administrator computer, such as, forexample, a keyboard, keypad, pointing device, mouse, touchpad,touchscreen, microphone, and/or other data entry means known in the art.User computers and administrator computers also may comprise an audiodisplay means (not shown in FIG. 13) such as one or more loudspeakersand/or other means known in the art for emitting an audibly perceptibleoutput. The configuration of user computers and administrator computersin a particular implementation of one or more systems of the presentdisclosure is left to the discretion of the practitioner.

System server 1304 may comprise one or more server computers, computingdevices, or systems of a type known in the art. System server 1304 maycomprise server memory. System server 1304 may comprise one or morecomponents of solid-state electronic memory, such as random accessmemory. System server 1304 may also comprise an electromagnetic memorysuch as one or more hard disk drives and/or one or more floppy diskdrives or magnetic tape drives, and may comprise an optical memory suchas a Compact Disk Read Only Memory (CD-ROM) drive. System server 1304may further comprise such software (operational and application),hardware, and componentry, as would occur to one of skill of the art,such as, for example, microprocessors, input/output devices, devicecontrollers, video display means, and the like.

System server 1304 may comprise one or more host servers, computingdevices, or computing systems configured and programmed to carry out thefunctions allocated to system server 1304. System server 1304 may beoperated by, or under the control of, a “system operator,” which may bean individual or a business entity. For purposes of clarity, Systemserver 1304 is shown in FIG. 13 and referred to herein as a singleserver. System server 1304 need not, however, be a single server. Systemserver 1304 may comprise a plurality of servers or other computingdevices or systems connected by hardware and software that collectivelyare operable to perform the functions allocated to the various systemsof present disclosure. Specifically, system server 1304 may be operableto be a web server, configured and programmed to carry out the functionsallocated to a system server according to the present disclosure.Further, although user computers 1302 and administrator computers 1306may be connected directly to system server 1304, these computers may beconnected to system server 1304 through any suitable network, such asnetwork 1308. Further, in one embodiment, the users need not be providedaccess to system server 1304, but instead the content posts from usersare made by the user(s) and saved to one or more particular locationsand the posts are accessed or harvested by the administrator or systemautomatically.

System server 1304 may be operably connected to the various usercomputers 1302 and/or an administrator computers 1306 by network 1308,which in an embodiment of the present disclosure comprises the Internet,a global computer network. However, network 1308 need not comprise theInternet. Network 1308 may comprise any means for electronicallyinterconnecting system server 1304 and a user computer 1302 and/or anadministrator computer 1306. Thus, it will be appreciated by those ofordinary skill in the art that the network 1308 may comprise theInternet, the commercial telephone network, one or more local areanetworks, one or more wide area networks, one or more wirelesscommunications networks, coaxial cable, fiber optic cable, twisted-paircable, the equivalents of any of the foregoing, or the combination ofany two or more of the foregoing. In an embodiment where system server1304 and user computer 1302 and/or an administrator computer 1306comprise a single computing device operable to perform the functionsdelegated to both system server 1304 and user computer 1302 and/or anadministrator computer 1306 according to the present disclosure, network1308 comprises the hardware and software means interconnecting systemserver 1304 and user computer 1302 and/or an administrator computer 1306within the single computing device. Network 1308 may comprise packetswitched facilities, such as the Internet, circuit switched facilities,such as the public switched telephone network, radio based facilities,such as a wireless network, etc.

The various systems, methods, schema, ontologies, and architectures ofthe present disclosure may be used for purposes outside of the medicaltranscription field as referenced in the various examples cited herein.For example, the system for analyzing verbal records may comprisevarious components and relationships suitable for use in any number ofareas where various experiences are utilized and processed, withfeedback being fed back into system componentry to improve overallsystem outcomes. In addition, various components described herein mayshare a name (or a portion thereof) but have duplicative referencenumbers, and therefore the descriptions for the various componentsshould read in view of one another.

In addition, and regarding the various systems of the presentdisclosure, such systems may be operable, as desired by a user of suchsystems, to generate visual, electronic (video, audio, database,transcript, etc.), and/or printed reports, outputs, outcomes, and thelike. Such exemplary outputs may be used for any number of purposes, andmay be useful generally to “report” results, data, and/or knowledgecontained within and generated from such systems. Furthermore, thedisclosure of the present application further encompasses uses of thevarious methods, systems, architectures, etc., to perform various tasksin connection therewith.

EXAMPLES Examples 1. α-Amylase Inhibition Saliva Screening Method

At least one assay used for the detection of α-amylases activity, asused herein includes the use of a chromagenic substrate,2-chloro-p-nitrophenol linked to maltotriose. Enzymatic activity ofα-amylase on the substrate yields 2-chloro-p-nitrophenol, which can bemeasured spectrophotometrically at 405 nm. The amount of α-amylaseactivity present in the experimental sample is directly proportional tothe increase in absorbance at 405 nm.

For the assay, test compounds are mixed with saliva, prior to theaddition of α-amylase substrate. The test compounds, such as GRASmaterials, are prepared at a concentration of 5,000 ppm in distilledwater. Following this preparation, 300 μl of α-amylase substrate(Salimetrics α-amylase Assay Kit) that has been pre-heated to 37° C. isadded to 20 μl of the test compound. To initiate the assay, 10 μl of adilute (3.5%) or full strength (100%) saliva solution is added to themixture. Following the initiation, each mixture is measured at 1 minuteintervals using a spectrophotometer (such as Molecular Devices M5reader) at 405 nm. Temperatures of the mixtures are kept constant at 37°C. during the assay.

2. α-Amylase Inhibitor Assays

A series of GRAS compounds were tested using the α-amylase assayprotocol described here. The tested compounds are included in Table I.From the screen of GRAS compounds, five compounds (CDI-030,034,036,037and 042) were shown to have α-Amylase Inhibitor Activity in dilute(3.5%) saliva (See FIG. 14). Further details of these five compounds mayalso be seen in Table 4.

TABLE 4 100% 0% Saliva Amylase Time CDI-30 CDI-34 CDI-36 CDI-37 CDI-42Control Control 0 min 1.60 0.40 3.63 4.00 0.80 4.00 0.09 2.03 0.36 3.203.85 1.13 4.00 0.09 2.02 0.40 3.46 4.00 0.90 4.00 0.09 1.87 0.45 4.004.00 0.84 4.00 0.10 % CV 10.67 9.13 9.42 1.85 16.35 0.00 1.84 Ave 1.880.40 3.57 3.96 0.92 4.00 0.09 % Std 46.96 10.04 89.29 99.08 22.93 100.002.34 % Inhibition 53.04 89.96 10.71 0.92 77.07 0.00 97.66 5 min 4.000.49 4.00 4.00 1.65 4.00 0.09 4.00 0.50 4.00 4.00 2.15 4.00 0.09 4.000.50 4.00 4.00 1.80 4.00 0.09 4.00 0.52 4.00 4.00 1.81 4.00 0.10 % CV0.00 2.32 0.00 0.00 11.31 0.00 2.79 Ave 4.00 0.50 4.00 4.00 1.85 4.000.09 % Std 100.00 12.61 100.00 100.00 46.24 100.00 2.36 % Inhibition0.00 87.39 0.00 0.00 53.76 0.00 97.64 15 min  4.00 0.53 4.00 4.00 2.374.00 0.09 4.00 0.61 4.00 4.00 2.38 4.00 0.10 4.00 0.54 4.00 4.00 2.254.00 0.09 4.00 0.56 4.00 4.00 2.17 4.00 0.10 % CV 0.00 6.67 0.00 0.004.33 0.00 1.88 Ave 4.00 0.56 4.00 4.00 2.29 4.00 0.09 % Std 100.00 14.02100.00 100.00 57.35 100.00 2.37 % Inhibition 0.00 85.98 0.00 0.00 42.650.00 97.64Among these five compounds, CDI-034 and CDI-42 were shown to inhibitα-Amylase in 100% saliva samples (See FIG. 15).

3. Lysozyme Inhibition Saliva Screening Method

At least one assay used for the detection of α-amylases activity, asused herein includes the use of a Micrococcus lysodeikticus labeled withfluorescein. The assay measures lysozyme activity on Micrococcuslysodeikticus cell walls, which are labeled to such a degree that thefluorescence is quenched. Lysozyme activity can relieve this quenching;yielding increased fluorescence that is proportional to lysozymeactivity.

For the assay, test compounds are mixed with saliva, prior to theaddition of lysozyme substrate. The test compounds, such as GRASmaterials, are prepared at a concentration of 5,000 ppm in distilledwater. Following this preparation, 50 μl of lysozyme substrate at 1mg/ml (Molecular Probes EnzChek Lysozyme Assay Kit) that has beenpre-heated to 37° C. is added to 50 μl of the test compound. To initiatethe assay, 50 μl of a dilute (3.5%) or full strength (100%) salivasolution is added to the mixture. Following the initiation, each mixtureis measured at 1 minute intervals using a spectrophotometer (such asMolecular Devices M5 reader) for absorption at 494 nm and fluorescenceemission at 518 nm. Temperatures of the mixtures are kept constant at37° C. during the assay.

4. Lysozyme Inhibitor Assays

A series of GRAS compounds were tested using the Lysozyme assay protocoldescribed above. The tested compounds are included in Table I. From thescreen of GRAS compounds, four compounds including Fixanal® Buffer 6.0(Sigma-Aldrich Co., CDI-030), bentonite (CDI-37), benzoic acid (CDI-40),and acetic acid (CDI-047) were shown to have Lysozyme Inhibitor Activityin dilute (3.5%) saliva (See FIG. 16). Further details of these fivecompounds may also be seen in Table 5.

TABLE 5 Cell Location RFU Reading (60 mins) CDI Candidate # 30 F 4 51.1337 E 5 92.06 40 H 5 35.82 47 G 6 33.53 CONTROLS Pooled Saliva E 12127.33 Fresh Saliva F 12 182.02 Lysozyme G 12 233.43Among these four compounds, benzoic acid (CDI-40), and acetic acid(CDI-047) were shown to inhibit Lysozyme in 100% saliva samples (datanot shown).

5. Galactose Oxidase Inhibition Saliva Screening Method

At least one assay used for the detection of galactose oxidase activity,as used herein, includes the use of a chromagenic or fluorogenicsubstrate, such as with Amplex® Red (Molecular Probes). In an embodimentof the assay used herein, galactose oxidase catalyzes the oxidation ofgalactose at the C6 position and generates hydrogen peroxide (H2O2). TheH2O2 then, in the presence of horseradish peroxidase (HRP), reacts with1:1 stoichiometry with Amplex® Red reagent to generate thered-fluorescent oxidation product, resorufin. Resorufin has absorptionand fluorescence emission maxima of approximately 571 nm and 585 nm,respectively, and because the extinction coefficient is high (54,000cm-1M-1), the assay can be performed either fluorometrically orspectrophotometrically.

For the assay, test compounds are mixed with Amplex Red, reactionbuffer, horseradish peroxidase (HRP) at 100 U/ml, and galactose stocksolution (according to manufacturers recommended protocol, MolecularProbes A22179), prior to the addition of saliva. The test compounds,such as GRAS materials, are prepared under various concentration fortesting. Following this preparation, 50 μl of the test compound solution(with Amplex Red, reaction buffer, and HRP) is added to 50 μl of saliva.Following the initiation, each mixture is measured at 1 minute intervalsusing a spectrophotometer (such as Molecular Devices M5 reader) usingexcitation in the range of 530-560 nm and emission detection at about590 nm or absorbance at 560 nm. Temperatures of the mixtures are keptconstant at 37° C. during the assay.

6. Galactose Oxidase Inhibitor Assays

A series of GRAS compounds were tested using the Galactose Oxidase assayprotocol described herein. The tested compounds are included in Table I.From the screen of GRAS compounds, four compounds (CDI-039, 040, 042,and 047) were shown to have Galactose Oxidase Inhibitor Activity indilute (3.5%) saliva (See FIG. 17). FIG. 17 includes the followingsymbols which are: open circle=Aluminum potassium sulfate dodecahydrate,open square=3-tert-butyl-hydroxyanisole, open triangle=benzoic acid,open diamond=acetic acid, filled circle=fresh saliva, and filledsquare=water (control blank).

7. Stabilization of Glycosylated Hemoglobin

At least one procedure used for the stabilization and detection ofglycosylated hemoglobin (HbA1c), as described herein, includes theincubation of HbA1c with a stabilizing agent prior to, or concurrentlywith, detection. Analysis of samples was conducted on a HPLC 1100 series(Agilent Technologies) with a 2.1X150 (3.5μ) C18 reverse phase column.The mobile phase used was acetonitrile with 6.5 mM Ammonium Carbonate. AHbA1c standard was prepared by dissolving HbA1c (Sigma Aldrich, 405 RM)with 1.0 ml of deionized water. The samples analyzed by HPLC includedpure HbA1c (FIG. 18), 100 ml HbA1c mixed with 100 ml of fresh saliva(FIG. 19), and 200 ml HbA1c mixed with 100 ml of fresh saliva and 100 mlof a stabilizing compound comprising aluminum sulfate hydrate, benzoicacid, and aluminum potassium sulfate dodecahydrate (each present in a1:1:1 ratio) (FIG. 20). While the pure HbA1c sample was analyzed fresh,samples of HbA1c with saliva were incubated overnight at roomtemperature prior to analysis. For each sample analyzed, the data wasdisplayed through a three dimensional plot (FIGS. 18A, 19A, and 20A), aUV image (FIGS. 18B, 19B, and 20B), and a chromatogram (FIGS. 18C, 19C,and 20C). Additionally, a comparison of the analysis HbA1c in thepresence of saliva with and without stabilizing agent is shown in FIG.21 (chromatography), 22 (UV image), and 23 (three dimensional plot).

8. HbA1c Protection Assays

A series of GRAS compounds were tested, as shown in FIGS. 18-20, for theability to protect HbA1c from degradation by components in saliva. Thetested compounds are included in Table I. As described above, astabilizing compound comprising aluminum sulfate hydrate, benzoic acid,and aluminum potassium sulfate dodecahydrate (each present in a 1:1:1ratio) minimized the degradation of HbA1c, and yielded a defined patternfor HbA1c.

9. Stabilization of Cancer Antigen 19-9

At least one procedure used for the stabilization and detection ofCancer Antigen 19-9 (CA 19-9) as described herein, includes theincubation of CA 19-9 with a stabilizing agent prior to, or concurrentlywith, detection. Analysis of samples was conducted on a HPLC 1100 series(Agilent Technologies) with a 2.1X150 (3.5μ) C18 reverse phase column.The mobile phase used was acetonitrile with 6.5 mM Ammonium Carbonate. ACA 19-9 standard was prepared by dissolving CA 19-9 with 1.0 ml ofdeionized water. The samples analyzed by HPLC included pure CA 19-9(FIG. 24), 100 ml CA 19-9 mixed with 100 ml of fresh saliva (FIG. 25),and 200 ml CA 19-9 mixed with 100 ml of fresh saliva and 100 ml of astabilizing compound comprising aluminum sulfate hydrate, benzoic acid,and aluminum potassium sulfate dodecahydrate (each present in a 1:1:1ratio) (FIG. 26). While the pure CA 19-9 sample was analyzed fresh,samples of CA 19-9 with saliva were incubated overnight at roomtemperature prior to analysis. For each sample analyzed, the data wasdisplayed through a three dimensional plot (FIGS. 24A, 25A, and 26A) anda UV image (FIGS. 24B, 25B, and 26B). Additionally, a comparison of theanalysis CA 19-9 in the presence of saliva with and without stabilizingagent is shown in FIG. 27 (three dimensional plot) and 28 (UV image).

10. CA 19-9 Protection Assays

A series of GRAS compounds were tested, as shown in FIGS. 24-26, for theability to protect CA 19-9 from degradation by components in saliva. Thetested compounds are included in Table I. As described above, astabilizing compound comprising aluminum sulfate hydrate, benzoic acid,and aluminum potassium sulfate dodecahydrate (each present in a 1:1:1ratio) minimized the degradation of CA 19-9 in at least one embodiment.

11. Peroxidase Assay

To determine the effect of various GRAS compounds as inhibitors ofperoxidase activity, a standard assay using Amplex® Red (LifeTechnologies), which releases a fluorescent oxidation product. Thisoxidation product, named resorufin, has an excitation and emissionmaxima of approximately 571 nm and 585 nm, respectively, and because theextinction coefficient is high (58,000±5,000 cm-1 M-1), it allows forthe use with fluorometric or spectrophotometric assays.

The test compounds, such as GRAS materials or cocktails of same, wereprepared at a concentration of 500 ppm in distilled water. For theassay, 50 μl samples of test compounds were mixed with 20-30 μl ofsaliva (either “pooled” or “unpooled”) prior to the addition of Amplex®Red. Following the mixture of the test compounds with saliva, 500 ofAmplex® Red/HRP working solution (See Protocol for Invitrogen Assay#A22188, which is incorporated herein in its entirety), which has aconcentration of 100 μM of Amplex® Red, is added to the testcompound/saliva mixture. Following the initiation of the reaction, eachmixture is measured at 1 minute intervals using a spectrophotmoter (suchas a Molecular Devices M5 reader) for excitation in the range of 530-560nm and fluorescence emission detection at approximately 590 nl, or forabsorption at approximately 560 nm. Reactions were allowed to run for 30minutes during these assays.

12. Peroxidase Protection Assays

A series of GRAS compounds and cocktails of GRAS compounds were tested,as shown in FIGS. 29-33, for the ability to inhibit degradation of aperoxidase-sensitive marker by components in saliva. Various embodimentsof the tested compounds are included in Table I. As described above, astabilizing compound/cocktail comprising aluminum sulfate hydrate,benzoic acid, and aluminum potassium sulfate dodecahydrate (each presentin a 1:1:1 ratio) minimized the degradation of a peroxidase sensitivemarker in at least one embodiment (FIG. 29, open circle=blank control,open square=stabilizing cocktail). Further, inhibition assays were alsoconducted with caffeine (FIG. 30, CDI #33; Key: open circle=pooledsaliva, open square=unpooled saliva, open triangle=caffeine, opentriangle=caffeine (repeat)), 3-tert-butyl-hydroxyanisole (FIG. 31, CDI#39; Key: open circle=pooled saliva, open square=unpooled saliva, opentriangle=3-tert-butyl-hydroxyanisole, opentriangle=3-tert-butyl-hydroxyanisole (repeat)), benzoic acid (FIG. 32,CDI #16; Key: open circle=pooled saliva, open square=unpooled saliva,open triangle=benzoic acid, open triangle=benzoic acid (repeat)), andaluminum potassium sulfate dodecahydrate (FIG. 33, CDI #42; Key: opencircle=pooled saliva, open square=unpooled saliva, opentriangle=aluminum potassium sulfate dodecahydrate, opentriangle=aluminum potassium sulfate dodecahydrate (repeat)), each ofwhich showed significant peroxidase inhibition capabilities.

13. Diagnostic Devices

In an example of a diagnostic device, as shown in FIG. 34, a test slidewith 28 test wells are spotted with 12 to 18 sets of diagnostic markers.These reagents may include diagnostic markers, or controls for thediagnosis. The diagnostic device, in this example, has four diagnosticmarkers spotted in duplicate, along with two positive and two negativecontrol marker. Each marker is spotted with a competitor, such asanti-haptin, which binds the capture antigen particle to the marker. Thedevice may have four different hatpin/anti-haptin systems to serve asthe generic capture system. A unique haptin may be conjugated to a fullyparamagnetic particle (PmMp). The PmMp is also coated with a capturemonoclonal antibody (MAb) specific for the marker. A second particle(polystyrene microparticle with Europium) is conjugated with the secondor detection MAb. In the diagnostic device shown in FIG. 34, the hashmarks on the side of the diagnostic device (slide) are indentationsmolded into the plastic, which may be used as cog grooves. Additionallythe slide is designed to fit into a processor that may have more thanone processing station, as shown in FIG. 36.

In performing a method of detecting a diagnostic marker using anembodiment of the diagnostic device, such as that in FIGS. 34 and 36,the user adds a pre-determined amount of specimen (such as 120 μl) to aspecimen diluent (such as 40 to 120 μl), and adds the mixture to eachtest well. Following addition of the diluted specimen, the test slide isincubated at 37° C. for about 5 to about 15 minutes with agitation.Following the incubation period, the test wells move over a magneticsource that brings the PmMp to the bottom of the well, so that there ismaximal binding of the PmMp with the anti-haptin bound in the test well.Next, specimen and assay reagents are removed, wash buffer is added andaspirated off. Lastly, each well is scanned using a visualizationdevice, such as a CCD camera, and then the fluorescent signal (or othersignal) of each marker is determined.

14. Fluid Collection Device

An example of a collection device for the collection of an bodily fluidsis depicted in FIG. 35. In this example, the collection device iscapable of collecting at least 500 μl of bodily fluid, such as saliva.The cap is first removed from the tube. Squeezing the bulb providessuction adequate to pull an amount of saliva into the bulb. In somecases, a filter may be included in the tube to remove unwanted materials(such as particulate and mucous). The filter may be of any known filtermaterial, such as rayon, that is appropriate for the purposes describedherein. Following the obtaining of the bodily fluid in the collectiondevice, a cap is placed on the tube, and a second cap is removed from adispensing tube. The dispensing tube is in fluid communication with thebulb and is sized and shaped to allow for drops of a predetermined sizeto be formed when the bulb is squeezed. For example, the dispensing tubemay allow drops of 30 μl to be formed when the bulb is squeezed.

While various embodiments of the systems, methods, and compositions ofthe present disclosure have been described in considerable detailherein, the embodiments are merely offered by way of non-limitingexamples of the disclosure described herein. It will therefore beunderstood that various changes and modifications may be made, andequivalents may be substituted for elements thereof, without departingfrom the scope of the disclosure. Indeed, this disclosure is notintended to be exhaustive or to limit the scope of the disclosure.

Further, in describing representative embodiments, the disclosure mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described.Other sequences of steps may be possible. Therefore, the particularorder of the steps disclosed herein should not be construed aslimitations of the present disclosure. In addition, disclosure directedto a method and/or process should not be limited to the performance oftheir steps in the order written. Such sequences may be varied and stillremain within the scope of the present disclosure.

1. A method for analyzing stabilization agents, the method comprisingthe steps of: introducing a diagnostic marker into each of a pluralityof detection sites of a detection platform, the detection platformcomprising: the plurality of detection sites each capable of receiving adiagnostic marker, a stabilization agent, and a detection agent; adetection device capable of determining a binding characteristic betweenthe detection agent and the diagnostic marker in the detection sites; acomputer database capable of receiving a plurality of bindingcharacteristics, the plurality of binding characteristics comprising atleast one binding property of a diagnostic marker to a detection agent;a processor operably coupled to the computer database and the detectiondevice, the processor having and executing a software programoperational to: determine the binding characteristic of the detectionagent to the diagnostic marker in each of the plurality of detectionsites; compare the binding characteristic among each of the plurality ofdetection sites to order the level of effect of the stabilizing agentsassayed on the binding characteristic between the detection agent andthe diagnostic agent; generate a binding record using the comparedbinding characteristics; and deliver the binding record to a recipient;introducing the stabilization agent to each of the plurality ofdetection sites containing the diagnostic marker; combining thedetection agent with the stabilization agent and diagnostic marker inthe detection sites; comparing the binding characteristic with theprocessor among each of the plurality of detection sites to order thelevel of effect of the stabilizing agents assayed on the bindingcharacteristic between the detection agent and the diagnostic agent;generating a binding report using the compared binding characteristics;and delivering the binding report to a recipient.
 2. The method of claim1, wherein the stabilizing agent is selected from the group consistingof a protease inhibitor, a DNase inhibitor, and a RNase inhibitor. 3.The method of claim 1, wherein the stabilizing agent prevents thedegradation or inactivation of the diagnostic marker for at least atwenty four hours period following interaction with the diagnosticagent.
 4. The method of claim 1, wherein the diagnostic marker isselected from the group consisting of a protein, a glycoprotein, anucleic acid, an enzyme, an enzyme inhibitor, and a metabolite.
 5. Themethod of claim 1, wherein the stabilizing agent is useful to completelyor substantially inactivate an enzyme selected from the group consistingof an amylase, a lysozyme, a peroxidase, a glycosidase, an esterase, aprotease, and a peptidase.
 6. The method of claim 1, wherein thestabilizing agent is selected from the group consisting of Fixanal®Buffer 6.0 (Sigma-Aldrich Co.), acetic acid, aluminum hydroxidebentonite, aluminum sulfate hydrate, aluminum potassium sulfatedodecahydrate, benzoic acid, caffeine, and 3-tert-butyl-hydroxyanisole,or a combination thereof.
 7. The method of claim 1, wherein thestabilizing agent comprises a plurality of stabilizing agents eachpresent in approximately the same concentration.
 8. The method of claim1, wherein the stabilizing agent is capable of inhibiting degradation ofthe diagnostic marker to an inhibitory degree, wherein the inhibitorydegree is selected from the group consisting of at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, and at least about 99%.
 9. The method of claim 1, wherein thestabilizing agent has a concentration selected from the group consistingof about 200 parts per million (ppm) to about 2000 ppm, about 400 ppm toabout 1600 ppm, about 600 ppm to about 1400 ppm, about 800 ppm to about1200 ppm, and about 400 ppm to about 600 ppm.
 10. The method of claim 1,wherein the stabilizing agent is able to inhibit the degradation orinactivation of the diagnostic marker for an inhibitory period selectedfrom the group consisting of at least one minute, at least about fiveminutes, at least about ten minutes, at least about fifteen minutes, atleast about thirty minutes, at least about one hour, at least about twohours, at least about four hours, and at least about eight hours. 11.The method of claim 1, wherein the diagnostic marker is selected fromthe group consisting of Aldose reductase, Angiogenin, Annexin A1, B-cellactivating factor (BAFF), B-cell lymphoma 2 (BCL2)-like 2, Beta Humanchorionic gonadotropin, Ca15-3, Calcyclin, Calvasculin, Cancer AntigenCA 19-9, Cancer Antigen CA 15-3, Cathepsin D, Caveolin-1, ChromograninA, Alpha-crystallin B chain (CRYAB), Endostatin, Eotaxin-2, Epithelialcell adhesion molecule (EpCAM), Ezrin, fatty acid binding protein 4(FABP4), Galectin-3, γ-glutamylcysteine ligase regulatory chain (GCLR),Gelsolin, Glucose 6-phosphate (G6P), Glycoprotein 130 (gp130),Glutathione S-transferase Mu 1 (GSTM1), Hepsin, High-mobility groupprotein B1 (HMGB-1), Insulin-like growth factor binding protein 1(IGFBP-1), Insulin-like growth factor binding protein 4 (IGFBP-4),Insulin-like growth factor binding protein 5 (IGFBP-5), Insulin-likegrowth factor binding protein 6 (IGFBP-6), LGL, latency associatedpeptide (LAP), macrophage stimulating protein (MSP), MHC class Ipolypeptide-related sequence A (MICA), Nucleoside diphosphate kinase B(NME2), Neuron-specific Enolase (NSE), Osteopontin, Osteoprotegerin,Pepsinogen, Peroxiredoxin, Phosphoserine aminotransferase (PSAT1),Prostate Specific Antigen, Receptor tyrosine-protein kinase erbB-3(ErbB3), Serpin B3, Vascular smooth muscle cell growth factor R2 (VSGFR2/KDR), Vascular endothelial growth factor R3 (VEGF R3/Flt-4),Thyroglobulin, Tyrosine kinase with immunoglobulin-like and EGF-likedomains 2 (TIE-2), Tissue plasminogen activator (tPA), Transforminggrowth factor beta (TGF-β1), Tumor necrosis factor receptor 1 (TNF-R1),urokinase-type Plasminogen Activator (uPA), urokinase-type PlasminogenActivator Receptor (uPAR), BrcaI, BrcaII, kallikreins, e-cadherin, Hoxpeptide, and Engrailed-2.